Expression of functional cytochrome P450 monooxygenase system in enterobacteria

ABSTRACT

A bacterial cell containing a functional cytochrome P450 monooxygenase system, said cell comprising a genetic construct capable of expressing a cytochrome P450 and genetic construct capable of expressing, separately from said cytochrome P450, a cytochrome P450 reductase, wherein each of the cytochrome P450 and the cytochrome P450 reductase have at their N-terminus a bacterial signal peptide. The cytochrome P450 and the cytochrome P450 reductase may be encoded by different constructs or the same construct. A bacterial cell containing a cytochrome P450 comprising a genetic construct encoding, and capable of expressing, said cytochrome P450 wherein the cytochrome P450 has, at is N-terminal, a bacterial signal peptide. The bacterial cells are useful as, for example, bioreactors, in drug testing and mutagenicity testing and as a source of cytochrome P450.

The present invention relates to the expression of cytochrome P450 inbacteria; in particular the invention relates to the expression of anactive eukaryotic cytochrome P450 enzyme system in bacteria,particularly Enterobacteria.

BACKGROUND AND PRIOR ART

Cytochrome P450 monooxygenases (P450s) form a superfamily ofhaemoproteins which catalyse the metabolism of a wide range ofcompounds. They catalyse the oxidation of lipophilic chemicals throughthe insertion of one atom of molecular oxygen into the substrate (Porter& Coon (1991) J. Biol. Chem. 261, 13469-13472). Mammalian P450s catalysethe metabolism of endogenous and exogenous compounds, includingsteroids, therapeutic drugs and carcinogens (Guengerich (1987) in“Enzymology of rat liver cytochromes P450”, ed. Guengerich, F. P., CRCPress, Boca Raton Fla., Vol. 1, pp 1-54; Guengerich & Shimada (1991)Chem. Res. Toxicol. 4, 391-407; Gonzalez (1992) Trends in Pharmacol.Sci. 12, 346-352). The different mammalian P450s exhibit a unique butoverlapping substrate specificity and display a high regio- andstereoselectivity (Crespi et al (1993) Toxicology 82, 89-104). Based ontheir major functions, mammalian P450s can be subdivided into two majorclasses: those involved primarily in the metabolism of steroids and bileacids, and those which mainly metabolise xenobiotics. Thexenobiotic-metabolising P450s are typically located in the endoplasmicreticulum of certain mammalian cells such as liver cells and are termedmicrosomal P450s.

The compounds metabolised by the latter group of P450s includetherapeutic drugs such as cyclosporin, nifedipine and debrisoquine aswell as carcinogens such as polycyclic aromatic hydrocarbons,nitrosamines and arylamines. To be catalytically active, microsomalP450s require a supply of electrons which are shuttled from NADPH viathe FMN and FAD prosthetic groups of NADPH-cytochrome P450oxidoreductase (P450 reductase; EC 1.6.2.4) (Smith et al (1994) Proc.Natl. Acad. Sci. USA 91, 8710-8714).

Comparison of the primary structures of P450s indicates that they arestructurally related to each other and are most likely derived from acommon ancestor. Based on their primary structure the P450s areclassified into families such as CYP1, CYP2 etc (Nelson et al (1996)Pharmacogenetics 6, 1-42).

Because of the importance of mammalian P450s in the metabolism oftherapeutic compounds and carcinogens, attempts have been made toexpress mammalian P450s in heterologous systems. For example, mammaliancells have been used to express P450s heterologously as described byDoehmer et al (1988) Proc. Natl. Acad. Sci. USA 85, 5769-5773; Aoyama etal (1990) Proc. Natl. Acad. Sci. USA 87, 4790-4793; and Crespi et al(1991) Carcinogenesis 12, 355-359.

Yeast cells have also been used for the heterologous expression ofcytochrome P450, for example by Renaud et al (1993) Toxicology 82, 39-52and Bligh et al (1992) Gene 110, 33-39.

More recently mammalian P450s have been expressed in Escherichia coli.

Gillam et al (1993) Arch. Biochem. Biophys. 305, 123-131 describes theexpression of modified human cytochrome P450 3A4 in E. coli andpurification and reconstitution of the enzyme.

Barnes et al (1991) Proc. Natl. Acad. Sci. USA 88, 5597-5601 describesthe expression and enzymatic activity of recombinant cytochrome P45017α-hydroxylase in E. coli.

Larson et al (1991) J. Biol. Chem. 266, 7321-7324 describes thatexpression of cytochrome P450 IIE1 lacking the hydrophobic NH₂-terminalsegment retains catalytic activity.

Shimada et al (1994) Carcinogenesis 15, 2523-2529 describes theactivation of procarcinogens by human cytochrome P450 enzymes expressedin E. coli.

Shet et al (1993) Proc. Natl. Acad. Sci. USA 90, 11748-11752 describesthe enzymatic properties of a purified recombinant fusion proteincontaining NADPH-P450 reductase.

Shet et al (1995) Arch. Biochem. Biophys. 318, 314-321 describes someproperties of a recombinant fusion protein containing the haem domain ofhuman P450 3A4 and the flavin domains of rat cytochrome P450 reductase.

Jenkins & Waterman (1994) J. Biol. Chem. 269, 27401-27408 describes thatflavodoxin and NADPH-flavodoxin reductase from E. coli support bovinecytochrome P450 c17 hydroxylase activities.

Fisher et al (1992) FASEB J. 6, 759-764 describes the expression ofhuman cytochrome P450 1A2 in E. coli.

Fisher et al (1992) Proc. Natl. Acad. Sci. USA 89, 10817-10821 describesthe expression in E. coli of fusion proteins containing the domains ofmammalian cytochromes P450 and NADPH-P450 reductase flavoprotein.

Chun & Chiang (1991) J. Biol. Chem. 266, 19186-19191 describes theexpression of cholesterol 7α-hydroxylase cytochrome P450 in E. coli.

Richardson et al (1995) Arch. Biochem. Biophys. 323, 87-96 describes theexpression of human and rabbit cytochrome P450s of the 2C subfamily inE. coli.

Gillam et al (1995) Arch. Biochem. Biophys. 319, 540-550 describes theexpression of cytochrome P450 2D6 in E. coli.

Dong & Porter (1996) Arch. Biochem. Biophys. 327, 254-259 describes astudy in which P450 reductase containing an N-terminal fusion to an ompAsignal peptide is co-expressed in E. coli with human P450 2E1 in whichthe second codon of the P450 (serine) is replaced with an alanine; noother changes to the P450 were made. In vivo activity with whole cellscould not be demonstrated.

WO 94/01568 describes the expression of P450_(17α)-hydroxylase in E.coli and also its fusion to a P450 reductase enzyme domain andexpression of the fusion protein in E. coli. P450 enzyme hybrids,incorporating the N-terminal nine amino acids from bovineP450_(17α)-hydroxylase are also disclosed.

U.S. Pat. No. 5,240,831 describes expression of P450_(17α)-hydroxylasein E. coli in a biologically active form without the need forco-expression or admixture of a cytochrome P450 reductase.

Gillam et al (1995) Arch. Biochem. Biophys. 317, 374-384 describes theexpression of cytochrome P450 3A5 in E. coli.

Gillam et al (1994) Arch. Biochem. Biophys. 312, 59-66, describes theexpression of modified human cytochrome P450 2E1 in E. coli.

Shet et al (1994) Arch. Biochem. Biophys. 311, 402-417 describes arecombinant fusion protein expressed in E. coli containing the domainsof bovine P450 17A and rat NADPH-P450 reductase.

Josephy et al (1995) Cancer Res. 55, 799-802 describes the bioactivationof aromatic amines by recombinant human cytochrome P450 1A2 expressed inSalmonella typhimurium.

Despite the extensive efforts to express an effective eukaryotic,particularly mammalian, P450 monooxygenase enzyme system in bacteria todate no system which is capable of metabolising compounds in whole cellshas been devised. This is particularly the case forxenobiotic-metabolising P450s which require a P450 reductase forenzymatic activity. More particularly, no bacterial cell system haspreviously been devised which allows cytochrome P450 and cytochrome P450reductase to form a functional cytochrome P450 monooxygenase system whenexpressed separately in the same bacterial cell. Similarly, despiteconsiderable efforts to produce bacteria which express a high level of aeukaryotic xenobiotic-metabolising P450, no satisfactory system has sofar been devised.

One object of the invention is to provide superior systems forexpressing P450s in a functional form in intact bacterial cells.

A further object of the invention is to provide an improved system forexpressing P450s whether or not with P450 reductase.

Bacterial systems which express a functional P450 enzyme system in wholecells are useful as “bioreactors” or they may find uses in drug-testingor carcinogen-testing systems or as biosensors or in environmentalremediation or in the production of hormones and so on. The expressionof eukaryotic P450s at high levels in bacteria provides a source of P450for structural studies.

SUMMARY OF INVENTION

A first aspect of the invention provides a bacterial cell containing afunctional cytochrome P450 monooxygenase system said cell comprising agenetic construct capable of expressing a cytochrome P450 and a geneticconstruct capable of expressing, separately from said cytochrome P450, acytochrome P450 reductase wherein the N-terminus of the cytochrome P450and the N-terminus of the cytochrome P450 reductase are each adapted toallow functional coupling of said cytochrome P450 and said cytochromeP450 reductase within said cell.

Preferably, the bacterial cell expresses a cytochrome P450 monooxygenasesystem which has a specific activity of at least 50 pmol/min/mg protein,more preferably at least 250 pmol/min/mg protein and still morepreferably at least 500 pmol/min/mg protein. These levels are measuredusing a suitable and effective substrate for the cytochrome P450monooxygenase system. Preferably the said preferred specific activitiesare of whole cells but the activities may also be those found infractions of the cells such as the membrane fraction.

Thus, a bacterial cell is provided that contains a functional cytochromeP450 monooxygenase system said cell comprising a genetic construct whichexpresses a cytochrome P450 and a genetic construct which expresses,separately from said cytochrome P450, a cytochrome P450 reductasewherein the said cytochrome P450 and the said cytochrome P450 reductasefunctionally couple within said cell.

By “cytochrome P450” we include any haem-containing polypeptide whichgives an absorption maximum in the region of 450 nm±5 nm in a reduced COdifference spectrum by virtue of the formation of a CO adduct of theFe(II) of said haem.

It is envisaged that the invention can be practised on any cytochromeP450.

Preferably, the cytochrome P450 is a eukaryotic cytochrome P450; morepreferably the cytochrome P450 is a mammalian cytochrome P450 and stillmore preferably the cytochrome P450 is a human cytochrome P450.

A large number of cytochrome P450 cDNAs or genes have been clonedincluding a large number of eukaryotic cytochrome P450 cDNA,particularly mammalian cytochrome P450 cDNAs. For example, Nelson et al.(1996) Pharmacogenetics 6, 1-42, incorporated herein by reference, liststhe known cytochrome P450 cDNA and genes and groups them into genefamilies and subfamilies based on the degree of sequence similarity and,to some extent, their chromosomal localization. Details of cytochromeP450 sequences are also available on the Internet.

These cytochrome P450 cDNAs and genes can readily be obtained usingcloning methods well known in the art, some of which are described belowand for example, described in Sambrook et al (1989) Molecular Cloning, alaboratory manual, Cold Spring Harbor Press, Cold Spring House, NewYork.

It is particularly preferred if the cytochrome P450 is a member of anyone of the cytochrome P450 CYP1, CYP2, CYP3 or CYP4 families. It isstill further preferred if the cytochrome P450 monooxygenase system isone which metabolises xenobiotics.

At least some members of the cytochrome P450 families CYP1, CYP2 andCYP3 are involved in the metabolism of xenobiotic compounds such astherapeutic drugs. Members of the cytochrome P450 CYP1 family areinvolved in the metabolism of, for example, caffeine, benzphetamine,phenacetin, theophylline, acetaminophen, antipyrine, 2-hydroxyestradiol,imipramine, tamoxifen and Zoxazolamine. Members of the cytochrome CYP2family are involved in the metabolism of, for example testosterone,aflatoxin, benzphetamine, cyclophosphamide, hexobarbital,6-aminochrysene, retinol, tolbutamide(methyl), phenytoin, S-warfarin,tienilic acid, diazepam, propanalol, amitryptyline, bufuralol,bupranolol, clozapine, codeine, debrisoquine, desipramine,dextromorphan, ethylmorphine, flecainide, haloperidol, lidocaine,nortryptilline, propanolol, sparteine, taxol, tetrahydrocannabinol,progesterone and mephenytoin.

Members of the cytochrome CYP3 family are involved in the metabolism of,for example, lovastatin, nifedipine, taxol, teniposide, testosterone,verapamil, vinblastine, vincristine, vindesine, benzphetamine, cortisol,cyclosporin A & G, diazepam, dihydroergotamine, estradiol,ethynylestradiol, imipramine and lidocaine.

More preferably the cytochrome P450 is any one of the P450s CYP3A4,CYP2D6, CYP2A6, CYP2E1, CYP2D9 and CYP2C9.

Conveniently, the cytochrome P450, apart from the adaptation to itsN-terminus, consists essentially of the same polypeptide sequence as thenative cytochrome P450. However, the term “cytochrome P450” specificallyincludes modifications to native cytochrome P450, for example,modifications which alter the length or other properties of anyhydrophobic N-terminal portion which may be present in the nativecytochrome P450 or modifications, such as single or multiple pointmutations or deletions, which modify the substrate specificity of thecytochrome P450 compared to the native cytochrome P450.

By “cytochrome P450 reductase” we include any NADPH-cytochrome P450oxidoreductase which is able to transfer electrons from NADPH tocytochrome P450. Mammalian cytochrome P450 reductase contains one eachof FMN and FAD prosthetic groups. It is preferred if the cytochrome P450reductase is derived from the same species as the cytochrome P450expressed in the bacterial cell. It is particularly preferred if thecytochrome P450 reductase is a mammalian cytochrome P450 reductase; rator human cytochrome P450 reductase are especially preferred. The humancytochrome P450 reductase CDNA is described in Smith et al (1994) Proc.Natl. Acad. Sci. USA 91, 8710-8714. The rat cytochrome P450 reductasecDNA is described in Porter et al (1990) Biochemistry 29, 9814-9818.

Cytochrome P450 reductase cDNAs and genes can readily be obtained usingcloning methods well known in the art, some of which are describedbelow.

Conveniently, apart from the adaptation to its N-terminus, thecytochrome P450 reductase consists essentially of the same polypeptidesequence of the native cytochrome P450 reductase. However, the term“cytochrome P450 reductase” specifically includes modifications tonative cytochrome P450, for example single or multiple point mutationsor deletions which modify the cofactor binding.

It is preferred that when the cytochrome P450 is a human cytochrome P450the cytochrome P450 reductase is human cytochrome P450 reductase.

By “functional cytochrome P450 monooxygenase system” we mean that oncethe cytochrome P450 and cytochrome P450 reductase are expressed in thebacterial cell the cell, by virtue of the presence of said cytochromeP450 and said cytochrome P450 reductase, is able to convert a substratefor the cytochrome P450 monooxygenase system into a product providedthat sufficient cofactors, such as NADPH and oxygen, are present.

By “functional coupling of said cytochrome P450 and said cytochrome P450reductase within the cell” we mean that the cytochrome P450 andcytochrome P450 reductase are juxtaposed within the cell so that thecytochrome P450 reductase can provide electrons, whether directly orindirectly, to the cytochrome P450 during catalysis. The adaptation tothe N-terminus of the cytochrome P450 or to the N-terminus of thecytochrome P450 reductase is merely one that allows functionalexpression and coupling of said cytochrome P450 and said cytochrome P450reductase within the cell. The adaptation may be one which aids thejuxtaposition of the cytochrome P450 and the cytochrome P450 reductase.

By “within the cell” we specifically include that the functionalcoupling may occur within or associated with any membrane or compartmentof the cell including the periplasmic space or any membrane associatedwith the periplasm.

It is preferred if, the cytochrome P450 content of a culture ofbacterial cells optimally expressing the cytochrome P450 is at least 100nmol/l culture of whole cells, preferably at least 150 nmol/l culture ofwhole cells more preferably almost 250 nmol/l culture of whole cells,still more preferably about 500 nmol/l culture of whole cells and mostpreferably about 1000 nmol/l. Typically the cytochrome P450 content isaround 200 nmol/l culture of whole cells.

Adaptions to each of the N-terminus of the cytochrome P450 andcytochrome P450 reductase which allow functional coupling of saidcytochrome P450 and said cytochrome P450 reductase are discussed below.

Although not essential, it is preferred if the cytochrome P450 orcytochrome P450 reductase retains its own N-terminal sequence and that afurther portion, such as a signal peptide as discussed below, is addedto the N-terminus. of the cytochrome P450 or cytochrome P450 reductaseor both.

Although it is envisaged that any aerobic or facultative anaerobicbacterial cell is suitable, it is preferred if the bacterial cell isGram-negative and more preferred if the bacterial cell is a cell of abacterium of the family Enterobacteriaceae, for example Escherichiacoli. The Enterobacteria most closely related to E. coli are from thegenus Salmonella and Shigella and less closely related are the generaEnterobacter, Serratia, Proteus and Erwinia. E. coli and S. typhimuriumare the most preferred bacterial host cells for the present invention.Strains of E. coli K12 are most preferred because E. coli K12 is astandard laboratory strain which is non-pathogenic.

Although certain cytochrome P450 substrates are able to penetrate intothe bacterial cell in order to be acted upon by the cytochrome P450monooxygenase system it is preferred if the bacterial cell of theinvention is one which has an increased permeability to a substrate orone which, when the bacterial cell is placed in an appropriate medium,becomes more permeable to the substrate. Additionally or alternativelyit is preferred if the bacterial cell is one which has altered membraneproperties, or a membrane whose properties can be altered, to facilitatethe membrane penetration of the substrates. For example, a tolC mutantof E. coli has a more permeable membrane than a wild type E. coli(Chatterjee (1995) Proc. Natl. Acad. Sci. USA 92, 8950-8954) and the TAseries of S. typhimurium strains have an increased permeability due to adeep rough mutation and have been frequently used for mutagenicitytesting (see for example Simula et al (1993) Carcinogenesis 14,1371-1376). S. typhimurium TA 97, 98, 100 and 102 as well as TA 1535 andTA 1538 have been used for mutagenicity testing in the pharmaceuticalindustry during drug safety evaluation. The present invention usingthese and other suitable bacterial strains are likely to improve theseprocedures, since they provide a humanized mutagenicity system and donot rely on rodent liver extracts (S9 fraction from rodent liver) as themetabolically activating system. The systems based on the presentinvention have also the advantage that the metabolically activatingsystem and the target for mutagenicity, namely the DNA are within thesame cell and are not physically separate entities as in the standardAmes test. This has the advantage that short lived metabolites arebetter detected and that the membrane barrier does not stop reactivemetabolites to reach their DNA target. This, and other aspects of theinvention, are discussed in further detail below.

It is also preferred if the cell is one which can be made more permeableby placing into the appropriate environment. Although there are manybuffers systems which are suitable it is preferred if, following theexpression phase, the bacterial cells, particularly E. coli cells, areresuspended in Tris-sucrose-EDTA, or TSE. TSE is 50 mM Tris. acetate (pH7.6), 0.25 M sucrose, 0.25 mM EDTA. This may increase the permeabilityof the cells in several ways. Firstly, the cells are initiallyresuspended in double strength buffer, and then diluted rapidly with anequal volume of water. This has the effect of causing the release ofperiplasmic proteins, by rupturing the outer membrane momentarily.Secondly, EDTA is known to affect permeability directly, and can rendercells more sensitive to certain hydrophobic agents. Thirdly, the Tris inthe buffer can affect the structure of the lipopolysaccharide in theouter membrane, again altering permeability. Further details of ways toincrease the permeability of the outer membrane of E. coli andSalmonella typhimurium is given in Nikaido & Vaara (1987) pp. 7-22 In:“Escherichia coli and Salmonella typhimurium. Cellular and MolecularBiology” Vol. 1. Ed. Neidhardt, F. C., Am. Soc. Microbiol., WashingtonD.C.

Advantageously, especially when the bacterial cells are used in abioreactor, the cells are solvent-resistant. Solvent-resistant E. colicells are known in the art for example from Ferrante et al (1995) Proc.Natl. Acad. Sci. USA 92, 7617-7621.

It is preferred if the cytochrome P450 reductase comprises an N-terminalportion which directs the cytochrome P450 reductase to a cellularcompartment or membrane of the bacterial cell. It is particularlypreferred if the said N-terminal portion directs the cytochrome P450reductase to a membrane.

It is preferred if the cytochrome P450 and the cytochrome P450 reductaseare associated with a membrane in the bacterial cell. It is particularlypreferred if the cytochrome P450 and the cytochrome P450 reductase areassociated with the bacterial inner membrane (particularly in the caseof E. coli and S. typhimurium), with their active sites located in thecytoplasm.

In one preferred embodiment the N-terminal portion is one which isderived from or based on an N-terminal portion of a bacterial proteinwherein said bacterial protein is one which is directed to theperiplasmic space or one which is destined for secretion from thebacterial cell. For example, the E. coli proteins encoded by the ompA,pelB, malE or phoA genes are such bacterial proteins. It is desirable ifthe presence of the N-terminal portion aids the correct folding of thecytochrome P450 or cytochrome P450 reductase.

Bacterial leader sequences or signal peptides which direct bacterialproteins usually into the periplasm have been fused previously to theN-terminus of a few mammalian proteins with a view to exporting theresulting fusion proteins to the oxidising environment of the periplasm.Thus, such bacterial leader sequences or signal peptides have been usedin the expression of mammalian secretory proteins such asimmunoglobulins or fragments thereof. In contrast, mammalianxenobiotic-metabolising cytochrome P450s, when found in the endoplasmicreticulum in nature, are usually exposed to a reducing environment.

Thus, a particularly preferred embodiment is wherein the N-terminalportion comprises any one of the ompA, pelB, malE or phoA signalpeptides or leader sequences or a functionally equivalent variantthereof.

By “functionally equivalent variant thereof” we include any peptidesequence which, if present in place in the native said bacterialprotein, would direct said protein to the same cellular location as thenatural signal peptide.

It is preferred if the N-terminal portion of each of the cytochrome P450and the cytochrome P450 reductase is a signal peptide or signal-peptidelike N-terminal portion.

It is particularly preferred if the N-terminal portion is one whichcompetes with the ompA leader for the general secretory pathway orcompetes with ompA for the signal recognition machinery, including thesignal recognition particle and trigger factor.

The general secretory pathway and components thereof are described inPugsley (1993) Microbiol. Rev 57, 50-108, incorporated herein byreference, and the signal recognition particle and trigger factor aredescribed in Valent et al (1995) EMBO J. 14, 5494-5505, incorporatedherein by reference. Competition assays between ompA signal peptide anda putative signal peptide can be carried out using methods known in theart using the teaching of Pugsley and Valent et al.

The pelB leader sequence consists of the amino acid sequenceMKYLLPTAAAGLLLLAAQPAMA (SEQ ID No 1).

The ompA leader sequence consists of the amino acid sequenceMKKTAIAIAVALAGFATVAQA (SEQ ID No 2).

Signal peptides have certain recognisable common features, which aredetailed in von Heijne (1986) Nucl Acids Res 14, 4683-4690; Gierasch(1989) Biochemistry 28, 923-930; and the chapter by Oliver (1987) onPeriplasm and Protein Secretion, pp. 56-69. Oliver (1987) In:“Escherichia coli and Salmonella typhimurium Cellular and MolecularBiology”, Vol. 1, Ed. Neidhardt, F. C., Am. Soc. Microbiol, WashingtonD.C. Firstly, there is an N-terminal net positively charged region ofvariable length (n-region). This is followed by a hydrophobic core(h-region), of 10±3 amino acids, which is rich in Leu, Ala, Met, Val,Ile, Phe and Trp residues. Finally, there is the c-region of typically5-7 amino-acids, which are generally slightly more polar than those inthe h-region. The most important amino acids in this c-region are thoseat the −3 and −1 positions relative to the site of signal cleavage (the“−3, −1 rule”) —there appear to be severe constraints on the possibleamino-acids which can exist in these positions: only those with smallside-chains are tolerated. Thus, only Ala, Gly, Leu, Ser, Thr and Valhave been found at −3 (with Ala strongly preferred), and only Ala, Gly,Ser and Thr at −1 (with Ala again strongly preferred). Evidence suggeststhat β-turn formation in this signal processing region is important forsignal cleavage to occur (see, for example, Barkocy-Gallagher et al(1994) J Biol Chem 269, 13609-13613, and Duffaud and Inouye (1988) JBiol Chem 263, 10224-10228.

It is preferred if signal peptide cleavage occurs. Thus, it is preferredif a suitable amino-acid sequence immediately downstream of the signalpeptide, ie. in the protein to be expressed is included, since thisstill forms part of the “cleavage site”. For example, a proline inposition +1 inhibits signal removal (Barkocy-Gallagher et al (1992) J.Biol. Chem. 267, 1231-1238). Using ompA as an example, it may be that toensure complete cleavage, if this is desirable, that the first fewamino-acids of the mature ompA protein in the construct, immediatelyafter the ompA leader, and immediately before the P450 or reductasesequence is included. Signal peptide cleavage is caused by a specificsignal peptidase enzyme, for example signal peptidase I. In a preferredembodiment signal peptidase I is overproduced in the bacterial cell (forexample, E. coli) to aid signal peptide cleavage if this is desirable(see van Dijl et al (1991) Mol. Gen. Genet. 227, 40-48).

It is particularly preferred if the first two amino acids of the matureOmpA protein (ie. Ala Pro) are inserted immediately downstream of theompA signal peptide and before the N-terminus of the P450.

Other preferred possibilities of increasing the probability of signalpeptide cleavage is to introduce a short linker sequence between thesignal peptide and the P450, or expression in different strains may leadto increased or reduced signal peptide cleavage compared with, forexample, expression in DH5α.

Thus it will be seen that it is preferred if the N-terminal portion is asignal peptide which when present in its natural polypeptide has thefunction to mediate the membrane insertion and the export of thenational polypeptide through the cytoplasmic membrane into theperiplasmic space. Sequence of the leader sequence or signal peptide isusually up to 40 amino acid residues. It is envisaged that leaders canbe modified without altering their functional properties.

In a further embodiment of the invention it is also preferred if thecytochrome P450 comprises an N-terminal portion which directs thecytochrome P450 to a cellular compartment or membrane of the bacterialcell. It is particularly preferred if the said N-terminal portiondirects the cytochrome P450 to a membrane.

The preferred N-terminal portions in this embodiment are the same as thepreferred N-terminal portions for the cytochrome P450 reductase. It isparticularly preferred if the N-terminal portion of the cytochrome P450comprises any one of the ompA, pelB, malE or phoA signal peptides orleader sequences, or a functionally equivalent variant thereof. The ompAsignal peptide is particularly preferred.

In a further particularly preferred embodiment the N-terminal portion ofthe cytochrome P450 and the N-terminal portion of the cytochrome P450reductase each direct the said cytochrome P450 or cytochrome P450reductase to the same cellular compartment or membrane. This isparticularly advantageous because it increases or improves thefunctional coupling of said cytochrome P450 and said cytochrome P450reductase within the cell. It is particularly preferred if thecytochrome P450 and cytochrome P450 reductase are directed to thecytoplasmic side of the inner membranes where access is gained to thebacterial cytoplasmic NADPH pool.

In further preference the N-terminal portion of the cytochrome P450 issubstantially the same as the N-terminal portion of the cytochrome P450reductase.

In a further embodiment the cytochrome P450 comprises an N-terminalportion which is adapted to increase the translatability or correctfolding of said cytochrome P450 in said bacterial cell.

Preferably, the cytochrome P450, compared to its native sequence, ismodified at the N-terminus thereof.

By “translatability” we mean the efficiency with which a given RNAmolecule can be translated into a polypeptide.

There are several features of the optimised CYP3A4 sequence whichimprove translatability.

These features include:

1. Second codon changed to suit E. coli preference (often GCT, encodingAla). This is demonstrated by Looman et al (1987) EMBO J 6, 2489-2492.

2. Codons 4 and 5 made rich in A and T residues (where possible), tominimise the potential for mRNA secondary structure around the startcodon. Minimisation of mRNA structure around the ribosome binding siteand start codon can have large effects on the “translatability” (see,for example, Wang et al (1995) Protein Expr Purif 6, 284-290.

Concerning the use of leader sequences, the principal advantage is thatby their nature, they are already “optimised” for bacterial expression,since they come from bacterial genes. This often includes the twofeatures described above for example, both pelB and ompA leaders containAAA (Lys) as the second codon, which was the best performing secondcodon in the paper of Looman et al in terms of “translatability”. Inaddition, they often have reduced secondary structure around theribosome binding site and start codon (see, for example, Movva et al(1980) J Mol Biol 143, 317-328 on ompA gene structure).

An additional advantage of using an N-terminal signal peptide fusionconcerns the minimisation of any effect of rare codons in the P450 orreductase cDNA. For example, the AGA/AGG codon is the least used in E.coli (Chen and Inouye (1990) Nucl Acids Res 18, 1465-1473), and slowsdown translation as a result of the limiting availability of thecorresponding charged tRNA molecule. However, the negative effect ofsuch a rare codon reduces as its distance from the start codon increases(Chen & Inouye, supra). By adding an “optimised” bacterial leadersequence (typically 20-25 amino-acids in length) to the N-terminus ofthe P450 (or reductase), any rare codon close to the 5′-end of the P450cDNA will be moved that much further away from the start codon, andtherefore have much less effect.

Thus, it is preferred if “translatability” is improved by one or more ofthe following means:

1. Removal of rare codons (see Chen & Inouye, supra, such as AGG/AGA(Arg), CUA (Leu), AUA (Ile), CCC (Pro), and GGA/GGG (Gly) from the P450or reductase cDNA, especially those less than 25-30 codons from thestart codon.

2. As an alternative to, or in conjunction with, the above, introductionof genes encoding the rare tRNA synthases, for example, the dnaY genefor AGG/AGA.

3. Making other changes to the DNA sequence to reflect E. colipreferences, for example, the non-random utilisation of codon pairs(Gutman & Hatfield (1989) Proc Natl Acad Sci USA 86, 3699-3703).

4. Making changes to the promoter/ribosome binding site within theexpression vector in order to minimise secondary structure potential andto optimise the distance between the ribosome binding site and the startcodon (see Wang et al (1995), supra).

It is also preferred if the cytochrome P450 has its N-terminus modifiedaccording to U.S. Pat. No. 5,240,831 or, for example, by the generalmethod of Gillam et al (1995) Arch. Biochem. Biophys. 317, 374-384, bothof which are incorporated herein by reference.

The bacterial cell of the invention comprises a genetic constructcapable of expressing a cytochrome P450 and a genetic construct capableof expressing a cytochrome P450 reductase.

Conveniently, the cell may contain one genetic construct capable ofexpressing both a cytochrome P450 and a cytochrome P450 reductase or thecytochrome P450 and the cytochrome P450 reductase may be expressed fromseparate genetic constructs.

The genetic construct may be DNA or RNA. DNA is preferred.

The genetic construct is typically an extrachromosomal genetic elementsuch as a plasmid or bacteriophage genome but the term “geneticconstruct” specifically includes that the genetic construct may be partof the bacterial chromosome. For example, the genetic construct may bepart of a bacteriophage which has lysogenised the bacterial chromosome.

The genetic construct comprises those genetic elements which arenecessary for expression of the cytochrome P450 or cytochrome P450reductase in the bacterial cell.

The elements required for transcription and translation in the bacterialcell include a promoter, a ribosome binding site, a coding region forthe cytochrome P450 or cytochrome P450 reductase.

In terms of the promoter, it is believed that virtually any promoterfunctional in the selected bacterial cell may be employed. However,preferred promoters include the lac, lac UV5, tac, trc, λP_(L), T7, lpp,lpp-lac or T3 promoter. Of course, the λP_(L), T7 and T3 promoters arederived from bacteriophage and are known to be functional in bacteriasuch as E. coli.

The use of a regulatable promoter, such as the lac promoter, ispreferred. It is preferred if a strong promoter, such as the T7promoter, is not used.

One will often desire to incorporate an appropriate ribosome bindingsite for effecting bacterial expression into the eukaryotic cytochromeP450 domain comprising gene. Often, the ribosome binding site andpromoter can be incorporated as a “cassette”, defined as a contiguous,pre-fabricated DNA segment which incorporates the desired elements andhas useful restriction enzyme recognition sites at its two termini,allowing it to be readily inserted at an appropriate point within thedesired cytochrome P450 gene or cDNA or cytochrome P450 reductase bysimple genetic manipulation.

Most conveniently, one may desire to simply employ a promoter andribosome binding site from a homologous system, such as the lac promoterand its associated RBS. In general, however, it is proposed that one mayemploy any effective bacterial ribosome binding site, with those RBSsfrom E. coli, λ, T7 or T3 being preferred. Even more preferred ribosomebinding sites are those from the T7 gene 10, or E. coli lac A, lac Z,trp A, trp B, trp C, trp D, trp E, trp L, trp R or trp S genes. Aparticularly preferred ribosome binding site and spacer region comprisesthe sequence 5′-AGGAGGTCAT-3′ (SEQ ID No 3), wherein the underlinedportion comprises the ribosome binding site and the adjacent CATsequence comprises the spacer region. (The spacer region is thatsequence between the ribosome site and the ATG initiation codon.)

One will also typically desire to incorporate an appropriate bacterialtranscription terminator, which functions to terminate the function ofbacterial RNA polymerases, the enzymes responsible for transcribing DNAinto RNA into a gene prepared in accordance with the invention. Therequirements for a functional bacterial transcription terminator arerather simple, and are usually characterized by a run of T residuespreceded by a GC rich dyad synunetrical region. The more preferredterminators are those from the TRP gene, the ribosomal terminators,rrnB, or terminator sequences from the T7 phage. In fact, the T7terminator sequences contain RNase III cleavage sites with a stem-loopstructure at the 3′ ends of mRNAs which apparently slows down messagedegradation.

The genetic construct is capable of propagation in the bacterial celland is stably transmitted to future generations.

A variety of methods have been developed to operably link DNA to vectorsvia complementary cohesive termini For instance, complementaryhomopolymer tracts can be added to the DNA segment to be inserted to thevector DNA. The vector and DNA segment are then joined by hydrogenbonding between the complementary homopolymeric tails to formrecombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. The DNAsegment, generated by endonuclease restriction digestion as describedearlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNApolymerase I, enzymes that remove protruding, 3′-single-stranded terminiwith their 3′-5′-exonucleolytic activities, and fill in recessed 3′-endswith their polymerizing activities.

The combination of these activities therefore generates blunt-ended DNAsegments. The blunt-ended segments are then incubated with a large molarexcess of linker molecules in the presence of an enzyme that is able tocatalyze the ligation of blunt-ended DNA molecules, such asbacteriophage T4 DNA ligase. Thus, the products of the reaction are DNAsegments carrying polymeric linker sequences at their ends. These DNAsegments are then cleaved with the appropriate restriction enzyme andligated to an expression vector that has been cleaved with an enzymethat produces termini compatible with those of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sitesare commercially available from a number of sources includingInternational Biotechnologies Inc, New Haven, Conn., USA.

A desirable way to modify the DNA encoding the polypeptide of theinvention is to use the polymerase chain reaction as disclosed by Saikiet al (1988) Science 239, 487-491.

In this method the DNA to be enzymatically amplified is flanked by twospecific oligonucleotide primers which themselves become incorporatedinto the amplified DNA. The said specific primers may containrestriction endonuclease recognition sites which can be used for cloninginto expression vectors using methods known in the art.

Suitably, the vectors include a prokaryotic replicon, such as the ColE1ori, for propagation in a prokaryote. The vectors can also include anappropriate promoter such as a prokaryotic promoter capable of directingthe expression (transcription and translation) of the genes in abacterial host cell, such as E. coli, transformed therewith.

A promoter is an expression control element formed by a DNA sequencethat permits binding of RNA polymerase and transcription to occur.Promoter sequences compatible with exemplary bacterial hosts aretypically provided in plasmid vectors containing convenient restrictionsites for insertion of a DNA segment of the present invention.

Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329available from Biorad Laboratories, (Richmond, Calif., USA) and pTrc99Aand pKK223-3 available from Pharmacia, Piscataway, N.J., USA.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well known methods that typicallydepend on the type of vector used. With regard to transformation ofbacterial host cells, see, for example, Cohen et al (1972) Proc. Natl.Acad. Sci. USA 69, 2110 and Sambrook et al (1989) Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.

Electroporation is also useful for transforming cells and is well knownin the art for transforming bacterial cells.

For example, many bacterial species may be transformed by the methodsdescribed in Luchansky et al (1988) Mol. Microbiol. 2, 637-646incorporated herein by reference. The greatest number of transformantsis consistently recovered following electroporation of the DNA-cellmixture suspended in 2.5× PEB using 6250 V per cm at 25 μFD.

Successfully transformed cells, ie cells that contain a DNA constructcapable of expressing said cytochrome P450 or cytochrome P450 reductase,can be identified by well known techniques. For example, cells resultingfrom the introduction of an expression construct of the presentinvention can be grown to produce the cytochrome P450 or cytochrome P450reductase. Cells can be harvested and lysed and their DNA contentexamined for the presence of the DNA using a method such as thatdescribed by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al(1985) Biotech. 3, 208. Alternatively, the presence of the protein inthe supernatant can be detected using antibodies as described below.

In addition to directly assaying for the presence of recombinant DNA,successful transformation can be confirmed by well known immunologicalmethods when the recombinant DNA is capable of directing the expressionof the protein. For example, cells successfully transformed with anexpression vector produce proteins displaying appropriate antigenicity.Samples of cells suspected of being transformed are harvested andassayed for the protein using suitable antibodies.

Thus, in addition to the transformed host cells themselves, the presentinvention also contemplates a culture of those cells, preferably amonoclonal (clonally homogeneous) culture, or a culture derived from amonoclonal culture, in a nutrient medium.

In a further preferred embodiment the bacterial cell further comprises agenetic construct capable of expressing a polypeptide cofactor whichaids the correct folding of the cytochrome P450 or the cytochrome P450reductase.

The presence of such a polypeptide cofactor is especially preferred whenexpressing the cytochrome P450 or the cytochrome P450 reductase from astrong promoter where, in the absence of the polypeptide cofactor, thecytochrome P450 or cytochrome P450 reductase may form non-functionalinclusion bodies.

Preferably, the polypeptide cofactor is a molecular chaperone such asthe Gro ELS complex, SecB, SecD, SecF, the DnaJ/DnaK/GrpE complex,peptidylprolyl-cis, trans isomerases, protein disulphide isomerase-likeproteins encoded by the genes dsbA, dsbB, dsbC and dsbD, the periplasmicchaperone encoded by clpB or thioredoxin. The solubility of foreignproteins expressed in E. coli has been increased by the coproduction ofbacterial thioredoxin (Yasukawa et al (1995) J. Biol. Chem. 270,25328-25331; incorporated herein by reference).

It may also be useful to use the following systems or host or expressionsystems.

1. The use of protease-deficient strains of E. coli (for example, ompT⁻,lon⁻, degP⁻), which may reduce degradation of the expressed protein(s).A set of E. coli strains deficient in all known loci affectingproteolytic of secreted recombinant proteins is described in Meerman &Georgion (1994) Biotechnology 12, 1107-1110.

2. The expression of the P450 and/or reductase as a fusion protein with,for example, ubiquitin (Baker et al (1994) J. Biol. Chem. 269,25381-25386), thioredoxin (eg pTrxFus vector from Invitrogen),glutathione S-transferase (eg pGEX vectors from Pharmacia) or protein A(eg pRIT2T vector from Pharmacia), any of which may enhance expression.

In a still further preferred embodiment the bacterial cell furthercomprises a genetic construct capable of expressing a polypeptidecofactor which aids transfer of electrons between the cytochrome P450and the cytochrome P450 reductase. Preferably, the said cofactor iscytochrome b₅ or the FMN domain of a cytochrome P450 reductase.

Other cofactors which may aid the transfer of electrons includeadrenodoxiniadrenodoxin reductase and NADH cytochrome b₅ reductase (inconjunction with cytochrome b₅). It is believed to be of particularbenefit to use a cofactor which takes electrons from NADH, rather thanNADPH in the present invention, or even to use both cofactors together,especially when dealing with whole cell metabolism (“bioreactors”). Thisis because the intracellular ratio of (NAD+NADH) to (NADP+NADPH) in E.coli is about 4:1. Therefore, there is a far greater potential for P450reduction (and therefore enzyme activity) if the reducing equivalent isNADH rather than NADPH. In conjunction with this, the invention includesadditions or modifications which may lead to an increase in thecytoplasmic pool of NADH and/or NADPH as a means of increasing enzymeactivity in whole cells. This includes addition of precursors to theextracellular medium (those for which an uptake mechanism exists, suchas nicotinamide), or inhibition of enzymes which destroy NADPH.

The FMN domain of cytochrome P450 reductase can be expressed asdescribed in Smith et al (1994) Proc. Natl. Acad. Sci. USA 91, 8710-8714and cytochrome b₅ can be expressed as described in Holmans et al (1994)Arch. Biochem. Biophys. 312, 554-565. It is preferred if a polypeptidecofactor which aids transfer of electrons is included when thecytochrome P450 is any one of CYP3A4, CYP3A5, CYP3A7, CYP2E1 and CYP1A1.It is particularly preferred if cytochrome b₅ is co-expressed with anyone of CYP3A4, CYP3A5, CYP3A7 or CYP2E1.

It is also particularly preferred if the FMN domain is co-expressed withCYP1A1.

Although it is envisaged that in some instances the said cofactor maycomprise an N-terminal portion which directs the cofactor to a cellularcompartment or membrane of the bacterial cell, it is preferred if nosuch modifications are made to cytochrome b₅ or the FMN domain ofcytochrome P450 reductase when they are expressed in the bacterial cell.

A further embodiment comprises a bacterial cell of the invention furthercomprising a genetic construct capable of expressing any one of anenzyme capable of metabolising the product of a reaction catalysed bythe cytochrome P450 monooxygenase system.

In order to attempt to mimic to metabolism of a compound by aeukaryotic, especially mammalian, cell or an organ from an animal,especially a mammal, it is desirable to express in the bacterial cell ofthe invention one or more further polypeptides which, in the eukaryoticcell or in the animal may metabolise further the product of thecytochrome P450 monooxygenase system. This is particularly beneficialwhen the bacterial cell of the invention is used for mutagenicitytesting or as a model for drug metabolism.

Conveniently, the enzyme is any of a glutathione S-transferase, anepoxide hydrolase or a UDP-glucuronosyl transferase. Other enzymesinclude sulfotransferase, N-acetyltransferase, alcohol dehydrogenase,γ-glutamyl transpeptidase, cysteine conjugate β-lyase,methyltransferase, thioltransferase, DT-diaphorase, quinone reductase orglyoxalase.

It will be appreciated that because several genetic constructs may bepresent in the same bacterial cell, for example constructs expressingcytochrome P450 and cytochrome P450 reductase and sometimes alsocytochrome b₅ or an FMN domain of cytochrome P450 reductase or a furtherenzyme, it is convenient if bacterial strains are provided which haveone or more of the genetic constructs integrated into their chromosomeand that these strains can then be used as a “master” strain for theintroduction of further genetic elements. For example, it isparticularly preferred if the bacterial “master” strains comprise acytochrome P450 reductase genetic construct integrated into thebacterial chromosome. It is also preferred if the bacterial “master”strains comprise a genetic construct or constructs which express bothcytochrome P450 reductase and cytochrome b₅ from the bacterialchromosome. These “master” strains are then transformed with a geneticconstruct capable of expressing a cytochrome P450.

A further aspect of the invention provides a method of culturing a cellof the first aspect of the invention. Any suitable culture medium may beused. It is preferred if a nutrient-rich broth, such as Terrific broth,is used. It is also preferred if the culture medium contains a compoundwhich aids haem synthesis; δ-amino levulinic acid (ALA) is particularlypreferred.

It will be appreciated that in all aspects of the invention where abacterial cell contains two or more genetic constructs those geneticconstructs are compatible with each other in the same bacterial cell. Ingeneral, genetic constructs which are integrated into the chromosome arecompatible with one another and two genetic constructs are usuallycompatible with one another when one is integrated into the chromosomeand the other is an autonomous replicon such as a plasmid. In general,when there are two or more different plasmids without the same cellwhich constitute the genetic constructs of the invention it is desirableif they are compatible plasmids, for example plasmids which havedifferent origins of replication. It is also desirable if the differentplasmids encode different antibiotic resistance genes so that all of thedifferent plasmids can be selected when the bacterial cell is grown inculture.

A second aspect of the invention provides a method of converting asubstrate for cytochrome P450 into a product, the method comprisingadmixing said substrate with a bacterial cell according to the firstaspect of the invention wherein said cell contains a functionalcytochrome P450 monooxygenase system which can convert said substrate.

A third aspect of the invention provides the use of a bacterial cellaccording to the first aspect of the invention for converting asubstrate of a cytochrome P450 into a product.

The bacterial cells of the invention will find uses in many fields oftechnology, particularly those cells of the first aspect of theinvention which express a functional cytochrome P450 monooxygenasesystem.

The following are some specific uses to which the bacterial cells of theinvention can be put but it is envisaged that there are many other uses,for example whenever it is desirable to convert a cytochrome P450substrate into a product.

a) Drug Development and Drug Testing

Bringing safe new drugs onto the market is expensive, complicated andprotracted. Efficacy and safety of the market product, with respect topharmacokinetic parameters, drug/drug interactions and toxicity, arecritically dependent on the models employed for drug development. Amajor advance in drug development would result if the shortcomings oflead compounds could be predicted at the earliest stage of development.

There are serious problems in extrapolating pharnacotoxicological datafrom animal models to man. These are often due to pronounced speciesdifferences in the catalytic properties of drug metabolizing enzymes,which determine the pharmacological and the toxicological properties ofmost therapeutic drugs.

The bacterial cells, particularly E. coli and S. typhimurium cells,which form part of the present invention and which express functionalP450 monooxygenase systems, are ideal models for mimicking human drugmetabolism and are easier to handle than yeast and mammalian cell basedmodels. These cells allow the high throughput screening of drugs withrespect to optimized drug metabolism properties. This issue becomesparticularly important with the advent of combinatorial chemicallibraries which necessitate the evaluation of the drug metabolismproperties of several hundred compounds within a short time.

In this embodiment it is useful if the bacterial cells also expressother drug-metabolizing enzymes as described herein.

b) Bioreactors

The bacterial cells of the invention, because of the high substrate,region and stereoselectivity of the oxidative reactions catalyzed byP450s are useful for the synthesis of fine or bulk chemicals and thesynthesis of intermediates of chemical reactions.

In this embodiment it is clear that a bacterial cell of the invention isselected which expresses a cytochrome P450 with the appropriatesubstrate specificity. The substrate specificity of many cytochromeP450s is known in the art and so the appropriate cytochrome P450 can bereadily selected. However, as more cytochrome P450 genes are found itwill be possible to use those in the invention and, indeed, thebacterial cells of the invention, which express a new cytochrome P450can be used to determine its substrate specificity.

It will be appreciated that because some cytochrome P450s are able toconvert alkanes to alcohols or aromatic compounds to phenolic compoundsthe bacterial cells of the invention are useful in the bulk chemicalindustry where such alcohols and phenolic compounds are required.However, many of the reactions catalysed by cytochrome P450s make thecells of the invention useful in the fine chemical and pharmaceuticalindustries where selective oxidation (including hydroxylation) ofcomplex structures is often required. It is believed that the cells ofthe invention are particularly suited to the synthesis of steroidhormones and analogues thereof.

c) Biocatalysis

The systems developed in the present invention will allow rapidfunctional testing of P450 variants generated by site directedmutagenesis. It will be therefore possible to generate within a shorttime novel P450s with improved catalytic properties.

d) Bio- and Chemo-sensors

The bacterial cells of the invention are also useful as bio- orchemo-sensors. In particular membranes isolated from the cells areuseful. Binding of substrates (which are the molecules to be sensed ordetected) can cause a change of potential when the bacterial cell or themembranes isolated from the cell are present on an electrode surfacethereby allowing detection of the substrate molecule. The use ofimmobilised cells for detection and analysis is described in Kambe &Nakanishi (1994) Current Opinion in Biotechnology 5, 54-59.

e) Bioremediation

The bacterial cells of the invention are also useful in bioremediation.For example, cytochrome P450 monooxygenase systems are able to detoxifyharmful compounds. The appropriate bacterial cells expressing anappropriate cytochrome P450 which can oxidise the harmful compound isuseful in rendering the said compound less harmless.

f) Carcinogenicity Testing

As is described in more detail else where, the cells of the invention,particularly S. typhimurium cells, are useful in carcinogenicitytesting.

Although it is envisaged that the cells of the invention are especiallyuseful because they provide a functional cytochrome P450 monooxygenasesystem within an intact cell, it is also part of the invention thatmembranes are isolated from said cells and that said membranes areenriched in the cytochrome P450 monooxygenase system compared with wholecells. Membrane isolation from bacterial cells is well known in the art.Membrane isolation from cells of the invention is described in moredetail in the Examples.

A fourth aspect of the invention provides a bacterial cell containing acytochrome P450 said cell comprising a genetic construct capable ofexpressing, said cytochrome P450 wherein the cytochrome P450 comprisesan N-terminal portion which directs the cytochrome P450 to a cellularcompartment or membrane of the bacterial cell.

It is preferred if the N-terminal portion directs the cytochrome P450 toa membrane.

It is further preferred if the N-terminal portion comprises the ompA,pelB, malE or phoA signal peptide or a functionally equivalent variantthereof.

The preferred features of the N-terminal portion, particularly those ofthe signal peptides or leader sequences, are those preferred in theprevious aspects of the invention.

It is still further preferred if the cytochrome P450 further comprises apeptide sequence which will aid purification of the cytochrome P450 fromthe bacterial cell; more preferably wherein said peptide sequencecomprises a binding site for a compound.

It is particularly preferred if said peptide sequence is a -(His-)_(n)where n ≧4 and said compound is nickel.

It is contemplated that the fourth aspect of the invention is usefulboth for those cytochrome P450s which ordinarily couple with cytochromeP450 reductase and for other cytochrome P450s such as those which coupleadrenodoxin/adrenodoxin reductase or equivalent electron transfercompounds. Thus, in a preferred embodiment of the fourth aspect of theinvention, the cell further comprises a genetic construct capable ofexpressing each, or both, of adrenodoxin or adrenodoxin reductase ofequivalent electron transfer components. By “equivalent electrontransfer components” we include all other functionally-equivalentcomponents which can transfer electrons from NADH to cytochrome P450,particularly those components whose natural function is to transferelectrons from NADH to particular cytochrome P450s.

A fifth aspect of the present invention provides a method of preparingcytochrome P450, the method comprising the steps of (a) providing asufficient quantity of cells according to the fourth aspect of theinvention and (b) separating the cytochrome P450 from the other cellularcompartments.

A further aspect of the invention provides a genetic construct capableof expressing a cytochrome P450 wherein the cytochrome P450 comprises anN-terminal portion which directs the cytochrome P450 to a cellularcompartment or membrane of a bacterial cell. The preferred features ofthe N-terminal portion are those preferred in relation to the otheraspects of the invention. It is particularly preferred if the N-terminalportion comprises the ompA, pelB, melE or phoA signal peptide or afunctionally equivalent variant thereof. Other preferred features of thegenetic construct are those preferred in the previous aspects of theinvention. A still further aspect of the invention provides a pluralityof bacterial cells of the first or fourth aspects of the invention, eachcell containing a genetic construct capable of expressing a differentcytochrome P450 or containing a genetic construct or constructs whichencode different combinations of cytochrome P450 and cytochrome P450reductase and, if appropriate, other polypeptides such as those whichaid electron transfer or those which will further metabolise the productof the reaction of the cytochrome P450 monooxygenase system with asubstrate.

Such a plurality (or library) of cells can conveniently be stored, forexample in suitable conditions in a freezer and, for example, in amicrotitre plate, each well containing a different bacterial cell. Theplurality of cells may be useful for drug-testing or carcinogenicitytesting and for other purposes such as those described above.

The invention is described in more detail below with reference to thefollowing Examples and Figures.

FIG. 1: Construction of pB216

Plasmid NF14 was modified by replacement of an existing transcriptionterminator by an annealed oligonucleotide pair consisting of a trpAterminator with SalI and BglII ends. This modification removed a secondBglII site, allowing subsequent cloning at the remaining BglII site, andcreating the plasmid pB215. A BclI-BglII fragment containingpelB-reductase with a P_(tac)P_(tac) promoter was subcloned from pB207into the BglII site of pB215, to make the co-expression plasmid pB216.The orientation of the pelB-reductase insert was found to be as shown.

FIG. 2: Western blot of membrane fractions containing reductase andCYP3A4 expressed in E. coli

10 μg of each membrane fraction was loaded onto each track. The order ofloading is shown along the top of the blot. Each expression sample isshown alongside its uninduced counterpart. The immunodetection wascarried out using antibodies against reductase and CYP3A. A human livermicrosome track (10 μg microsomal protein) was included as a referencetrack.

FIG. 3: A representation of the two expression vectors used in thisstudy.

Plasmid pCW (FIG. 3A) contains a colel origin of replication and thebeta-lactamase gene, giving resistance to antibiotics such as ampicillinand carbenicillin. It was used for the expression of the P450 cDNAs.Plasmid pACYC184 (FIG. 3B) contains a p15A origin of replication, andwas used for expression of the P450 reductase cDNA, when a P450 cDNA wasbeing concomitantly expressed from pCW. The antibiotic used forselection of pACYC 184 was chloramphenicol. Unique restriction sites areshown in bold.

FIG. 4: Western blot showing expression of P450 reductase in bacterialmembranes.

Proteins were separated by SDS-PAGE, transferred onto nitrocellulosemembrane, and then probed with rabbit anti-reductase primary antibodyand horseradish peroxidase-linked anti-rabbit IgG secondary antibody.Detection was by chemiluminescence. Protein loading was 10 μg per track.Expression of the wild-type P450 reductase cDNA under non-inducing andinducing conditions is shown in tracks 1 and 2, respectively.Corresponding expression of a bacterial pelB leader-P450 reductaseN-terminal fusion protein is shown in tracks 3 and 4. A sample of humanliver microsomes is shown in track 5 for comparison. Reductaseactivities (in nmol cytochrome c reduced per min per mg protein) aregiven below the figure.

FIG. 5: Western blot showing the expression of pelB- and ompA-CY3A4 inbacterial membranes.

Proteins were separated by SDS-PAGE on a 9% acrylamide gel, transferredonto nitrocellulose, and probed with rabbit anti-CYP3A primary antibodyand horseradish peroxidase-linked anti-rabbit IgG secondary antibody.Detection was by chemiluminescence. Lanes 2 and 3 contain membranesisolated from bacteria expressing either pelB-CYP3A4 or ompA-CYP3A4,respectively (24 μg protein per track). Lane 1 contains a sample ofhuman liver microsomes for comparison (8 μg protein).

FIG. 6: Reduced Fe-CO spectra obtained from whole bacteria expressingpelB-CYP3A4 (FIG. 6A) ompA-CYP3A4 (FIG. 6B), or membranes derived fromthese cells.

Aliquots of cells or membranes were diluted 1:20 into 100 mM Tris-HCl,pH 7.4, containing 20% glycerol, 10 mM CHAPS and 1 mM EDTA, reduced bythe addition of a few crystals of sodium dithionite, and then dividedequally between a pair of matched glass cuvettes. Followingdetermination of a baseline spectrum over the range 500 to 400 nm, thesample cuvette was bubbled gently with CO for about 1 minute. The scanwas then repeated, and P450 content estimated using an extinctioncoefficient of 91 mM⁻¹ cm⁻¹.

FIG. 7: Summary of the expression levels of CYP3A4 from differentconstructs in E. coli, as estimated from the reduced Fe-CO differencespectra (see above).

Results are expressed as mean±SD, with tie number of determinations inparentheses. Cells were grown under standardised conditions (TerrificBroth, 30° C., 24 h induction)±0.5 mM δ-ALA.

FIG. 8: Western blot showing the expression of ompA-CYP2D6 in bacterialmembranes.

Proteins were separated by SDS-PAGE on a 9% acrylamide gel, transferredonto nitrocellulose, and then probed with a mixture of rabbitanti-CYP2D6 and anti-reductase primary antibodies, followed byhorseradish peroxidase-linked anti-rabbit IgG secondary antibodies.Detection was by chemiluminescence. Tracks 1 to 4 each contain 2.5 μgbacterial membrane protein. Track 5 contains 10 μg human livermicrosomes, and track 6 protein standards. Membranes were isolated fromcells carrying the empty expression vector, pCW (lane 1), ompA-2D6 alone(lane 2), or ompA-2D6 plus P450 reductase, cultured either in theabsence (lane 3) or presence (lane 4) of the haem precursordelta-aminolevulimc acid.

FIG. 9: Reduced Fe-CO spectra obtained from whole bacteria expressingompA-CYP2D6 alone (FIG. 3A), ompA-CYP2D6 co-expressed with P450reductase (FIG. 3B), or membranes derived from these cells.

Aliquots of cells or membranes were diluted 1:20 into 100 mM Tris-HCl,pH 7.4, containing 20% glycerol, 10 mM CHAPS and 1 mM EDTA, reduced bythe addition of a few crystals of sodium dithionite, and then dividedequally between a pair of matched glass cuvettes. Followingdetermination of a baseline spectrum between 500 and 400 nm, the samplecuvette was bubbled gently with CO for about 1 min. The scan was thenrepeated, and P450 content estimated using an extinction coefficient of91 mM⁻¹, cm⁻¹.

FIG. 10: Summary of the observed levels of ompA-CYP2D6 when expressedalone or co-expressed with P450 reductase in E. coli, as estimated fromthe reduced Fe-CO spectra (see above).

Results are expressed as mean±SD, with the number of determinations inparentheses. Cells were grown under standardised conditions (TerrificBroth, 30° C., 24 h induction)±0.5 mM δ-ALA.

FIG. 11: Kinetic parameters determined for the 1′-hydroxylation ofbufuralol in bacterial membranes co-expressing ompA-CYP2D6 and P450reductase.

Membranes were incubated at 37° C. with varying concentrations ofsubstrate (0-100 μM) in the presence of a NADPH generating system, underconditions which gave linear product formation with respect to proteinand time (not shown). The extent of product formation was assessed byreversed-phase HPLC, with reference to authentic standard. Kineticparameters were estimated from a double-reciprocal plot of initialvelocity against substrate concentration.

FIG. 12: P450 yield achieved in E. coli using the gene fusion strategyin comparison to the yield obtained by others after modifying the P450N-terminus by deletions and mutations.

FIG. 13: Reduced Fe-CO spectra obtained from whole bacterial expressingompA-CYP2A6, or from membranes derived from these cells.

Aliquots of cells or membranes were diluted 1:20 into 100 mM Tris-HCl,pH 7.4, containing 20% glycerol, 10 mM CHAPS and 1 mM EDTA, reduced bythe addition of a few crystals of sodium dithionite, and then dividedequally between a pair of matched glass cuvettes. Followingdetermination of a baseline spectrum between 500 and 400 nm, the samplecuvette was bubbled gently with CO for 1 min. The scan was repeated, andP450 content estimated using an extinction coefficient of 91 mM⁻¹ cm⁻¹.

FIG. 14: Summary of the observed levels of ompA-CYP2A6 when expressed inE. coli, as estimated from the reduced Fe-CO spectra (see above).

Results are expressed as mean±SD, n=3. Cells were grown understandardised conditions (Terrific Broth, 30° C., 24 h induction) in thepresence of δ-aminolevulinic acid (0.5 mM).

FIG. 15: Western blot showing the expression of ompA-CYP2E1 in bacterialmembranes.

Proteins were separated by SDS-PAGE on a 9% acrylamide gel, transferredonto nitrocellulose, and probed with sheep anti-CYP2E1 primary antibodyand horseradish peroxidase-linked anti-sheep IgG secondary antibody.Detection was by chemiluminescence. Lane 2 contains membranes isolatedfrom control bacteria harbouring the empty expression plasmid, pCW, andlane 3 membranes isolated from bacteria expressing ompA-CYP2E1 (24 μgprotein per track in each case). Lane 1 contains a sample of human livermicrosomes for comparison (8 μg protein).

FIG. 16: Reduced Fe-CO spectra obtained from whole bacteria expressingompA-CYP2E1.

Aliquots of cells were diluted 1:20 into 100 mM Tris-HCl, pH 7.4,containing 20% glycerol, 10 mM CHAPS and 1 mM EDTA, reduced by theaddition of a few crystals of sodium dithionite, and then dividedequally between a pair of matched glass cuvettes. Followingdetermination of a baseline scan from 500 to 400 nm, the sample wasbubbled gently with CO for 1 min. The scan was repeated, and P450content estimated using an extinction coefficient of 91 mM⁻¹ cm⁻¹.

Expression level: 451 nmol/l culture in whole cells.

FIG. 17: Metabolism of nifedipine by CYP3A4 in intact JM109, AB1157,NS3678 and TA1535 [pB2161] cells

Incubations were carried out in M9 salts supplemented with glucose (10mM) at 37° C. with shaking (200 rpm). 0.5 ml of 10× concentrated cellswas added to 4.5 ml of buffer and pre-equilibrated at 37° C. for 5-10min before addition of nifedipine to a final concentration of 200 μM. Atintervals 200 μl aliquots were removed and processed for HPLC analysisas described in Materials and Methods, in Example 4.

FIG. 18: Metabolism of testosterone by CYP3A4 in intact JM109, AB1157,NS3678 and TA1535 [pB216] cells

Incubations were carried out in M9 salts supplemented with glucose (10mM) at 37° C. with shaking (200 rpm). 0.5 ml of 10× concentrated cellswas added to 4.5 ml of buffer and pre-equilibrated at 37° C. for 5-10min before addition of testosterone to a final concentration of 200 μM.At intervals 200 μl aliquots were removed and processed for HPLCanalysis as described in Materials and Methods, in Example 4.

EXAMPLE 1

Materials and Methods

Bacterial Strains and Plasmids

Co-expression was compared in the E. coli K12 strains JM109 (Yanisch etal (1985) Gene 33, 103-19) and DH5α (Woodcock et al (1989) Nucleic AcidsRes 17, 3469-78), and the strain selected and used throughout was JM109.The vector pCW was used for the expression of reductase, as this haspreviously been used successfully for expression of several mammalianP450 cDNAs, including CYP3A4 (Gillam et al (1993) Arch. Biochem.Biophys. 305, 123-131). The plasmid pB207 comprises the pCW vectorcontaining the human reductase cDNA translationally fused to thebacterial pelB signal sequence. The sequence of the 5′ end of the cDNAis:

[ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATGGATATCGGATCCGAATT CCGCAACATG-human reductasecDNA (˜2 kb)] (SEQ ID No 4),

where the pelB leader sequence is shown underlined and the nativereductase ATG is shown in bold. NF14 (pCW containing an optimised CYP3A4sequence) was constructed in such a way as to be identical to that ofGillam et al (Gillam et al (1993) Arch. Biochem. Biophys. 305, 123-131).Plasmid pB215 was constructed by replacing the SalI-BglII fragment ofNF14 that contains a transcription terminator with a SalI-BglIIdouble-stranded oligonucleotide containing a trpA transcriptionterminator of the following sequence (top strand only shown):TCGACAGCCCGCCTAATGAGCGGGCTTTTTTTTA (SEQ ID No 5). A BclI-BglII fragmentfrom pB207 containing the pelB-reductase cDNA with its expression signalwas subcloned into pB215 at the unique BglII site to create pB216.

Co-expression of CYP3A4 and Reductase in E. coli

Expression conditions were a modification of those described previously(Gillam et al (1993) Arch. Biochem. Biophys. 305, 123-131). JM109 cellswere transformed with pB216 as described (Inoue et al (1990) Gene 96,23-8) and transformants isolated on LB agar plates containing 50 μg/mlampicillin. Single colonies were prepared, and were used to inoculate 5ml starter cultures in LB broth containing ampicillin, which were grownwith shaking at 37° C. overnight. Expression cultures were generally 100ml cultures (in 1 l flasks) containing Terrific Broth modified andsupplemented as described (Gillam et al (1993) Arch. Biochem. Biophys.305, 123-131). Expression cultures were inoculated with 1 ml overnightculture and grown shaking at 200 rpm, 30° C. for 4-5 h prior toinduction with 1 mM IPTG and addition of 0.5 mM δ-aminolevulinic acid.Growth and expression of heterologous proteins was then continued for20-24 h at 30° C., with 200 rpm shaking.

Harvesting Cultures and Determination of Expression Levels

Cells were harvested and resuspended in 5 ml 100 mM Tris. acetate (pH7.6), 0.5 M sucrose, 0.5 mM EDTA (2×TSE) as described (Gillam et al(1993) Arch. Biochem. Biophys. 305, 123-131). An equal volume ofice-cold distilled water was then added. The CYP3A4 content wasdetermined using Fe²⁺—CO vs Fe²⁺ difference spectra by adding 50 μlwhole cell suspension in 1×TSE to 950 μl 100 mM Tris.Cl (pH 7.4), 10 mMCHAPS, 20% v/v glycerol, 1 mM EDTA and reducing by adding a few grainsof sodium dithionite. A baseline of zero absorbance was recorded between500 and 400 nm, then the sample cuvette was bubbled under a steadystream of CO for about 1 minute. A spectrum was then recorded and theyield of spectrally active CYP3A4/l culture determined. At this stage,an aliquot of whole cells was withdrawn for metabolism studies. Membranefractions were isolated from the remaining cells as described (Gillam etal (1993) Arch. Biochem. Biophys. 305, 123-131). The CYP3A4 andreductase content of the membranes was determined. The yield of activereductase was evaluated by a spectrophotometric assay, as follows. To990 μl 50 μM cytochrome c in 0.3 M potassium phosphate buffer (pH 7.7),1-10 μg membrane protein was added, and a baseline recorded. 50 μM NADPHwas added and ΔA_(550nm) was recorded over time. To determine the P450contents of membranes, spectra were obtained as for whole cells.

Immunodetection of Heterologously Expressed CYP3A4 and Reductase

10 μg membrane protein was loaded per track onto a SDS-9% polyacrylamidegel, and the proteins separated by electrophoresis. Proteins were thentransferred to an ECL-nitrocellulose membrane (Amersham), blocked with10% milk powder in TBS-T [50 mM Tris. Cl (pH 7.9), 150 mM NaCl, 0.05%Tween-20] for 20 minutes, then incubated with diluted primary antibodiesfor up to 1 h (a mixture of rabbit anti-CYP3A and anti-reductaseimmunoglobulins were used). The membrane was then washed in TBS-T, andincubated in diluted HRP-linked donkey anti-rabbit antisera for about 45minutes. After washing, reductase and CYP3A4 were detected using ECLreagents (Amersham). Anti-reductase and anti-CYP3A4 antisera wereprovided by the ICRF Clare Hall facility, whereas HRP-linked donkeyanti-rabbit antibodies were obtained from the Scottish AntibodyProduction Unit.

Testosterone 6β-hydroxylase Assays

Assays were carried out with cells and membrane fractions. In bothcases, approximately 100 pmol P450 was incubated with shaking in TSEcontaining 30 mM MgCl₂. The final testosterone concentration was 0.2 mM.Where membranes were used, an NADPH generating system was added (finalconcentration 1 mM NADP, 5 mM glucose-6-phosphate, 1 unitglucose-6-phosphate dehydrogenase). Reactions were carried out at 37° C.for 5 minutes, then stopped by addition of 1 ml ice-cold methanol andplaced on ice for 10 minutes. Following centrifugation, supernatantswere diluted with an equal volume of ice-cold water, and thetestosterone metabolites extracted using Isolute C18 columns (IST Ltd),and eluted in 1 ml methanol. The methanol was evaporated in a SpeedVac,and the metabolites then resuspended in 200 μl 35% v/v methanol, andtransferred to HPLC vials. Metabolites were separated by HPLC on aSpherisorb ODS-2 (5 μm) 250×4.6 mm column using a gradient based onwater, methanol and acetonitrile, at a flow rate of 1 ml/min, anddetected at 240 nm. The yield of the 6β-hydroxytestosterone wascalculated by reference to a standard of known concentration, and thisallowed determination of the specific activity of the recombinant CYP3A4towards testosterone. The HPLC method was supplied by Glaxo-Welicome,and testosterone metabolites by Steraloids Inc. (a gift from SterlingWinthrop).

Erythromycin N-demethylase Assays

Bacterial membrane fractions were incubated with 0.5 mM erythromycin in50 mM HEPES buffer (pH 7.5) containing 150 mM KCl and 10 mM MgCl₂ in thepresence of NADPH generating system (as above), for 20 minutes at 37° C.The reaction was then stopped by the addition of 7.5% w/vtrichloroacetic acid, and the protein precipitate collected bycentrifugation. The erythromycin N-demethylase activity was thendetermined by a spectrophotometric assay performed using Nash reagent[6M ammonium acetate, 60 mM acetyl acetone, 150 mM acetic acid; (Nash(1953) Biochem. J 55, 416-421], measuring A_(412nm).

Nifedipine Oxidase Assays

Cells or membrane fractions were incubated with 0.2 mM nifedipine in TSEcontaining 30 mM MgCl₂ at 37° C. for 10 minutes with shaking. Wheremembrane fractions were used, NADPH generating system was included (asabove). The reactions were stopped by adding ice-cold methanol (30% v/vfinal concentration) and perchloric acid (1.5 v/v final concentration)and the precipitated protein collected by centrifugation. Thesupernatants were transferred to HPLC vials and nifedipine and itsoxidised metabolite were separated isocratically on a Spherisorb ODS-2(5 μm) 250×4.6 mm column using a mobile phase of methanol, acetonitrileand water (25:30:45 by volume), and detected at 254 nm by HPLC. Theamount of product formed was calculated by reference to a standardcontaining a known concentration of oxidised nifedipine.

Results

Construction of a Plasmid for Co-expression of CYP3A4 and Reductase

Preliminary experiments to optimise expression of reductase in E. coliindicated that high levels of controllable expression were achieved whenthe human reductase cDNA was translationally fused at its N-terminal tothe bacterial pelB leader sequence and expressed from the P_(tac)P_(tac)promoter of pCW, in the plasmid designated pB207; these expressionlevels were substantially higher than those obtained from the comparableconstruct lacking the pelB leader (data not shown). These results areconsistent with the previous expression of rat reductase fused toanother bacterial signal sequence, ompA (Shen et al (1989) J. Biol.Chem. 264, 7584-7589). A co-expression plasmid was constructed bysubcloning the pelB-reductase cDNA into the optimised CYP3A4 expressionplasmid NF14 (Gillam et al (1993) Arch. Biochem. Biophys. 305, 123-131)(reconstructed for the present work) as follows. NF14 was modified byremoving one of the BglII restriction sites within the vector, therebyallowing the downstream BglII site to be used for the subsequent cloningof reductase while retaining the terminator of CYP3A4 expression (seeMethods and Materials and FIG. 1 for details; pB215). A BclI-BglIIfragment from pB207 containing the pelB-reductase cDNA and itsP_(tac)P_(tac) promoter was then subcloned into pB215 at the BglII site,creating a plasmid, pB216, in which the two cDNAs, each bearing theP_(tac)P_(tac) promoter, were arranged head-to-tail (see FIG. 1).

Optimisation of Co-expression of Reductase and CYP3A4 from pB216

The ideal culture conditions were found to be similar to thoseestablished to be optimal for expression of CYP3A4 from NF14 (Gillam etal (1993) Arch. Biochem. Biophys. 305, 123-131), except thatsupplementation of cultures with δ-aminolevulinic acid was found tosubstantially increase expression of CYP3A4 (unpublished data), and istherefore now routinely used. Expression levels in the E. coli strainsJM109 and DH5α were compared, and it was found that while the level ofexpression of reductase was slightly higher in DH5α than in JM109, thiswas at the expense of the CYP3A4 level (data not shown). JM109 wasconsequently selected as the expression strain, as it was decided thatthe level of CYP3A4 expression should be considered the priority.

Previous reports of expression of reductase in E. coli indicated thatthe growth medium was supplemented with riboflavin (Shen et al (1989) J.Biol. Chem. 264, 7584-7589). We found that the addition of riboflavinmade negligible difference to the level of expression of activereductase, so riboflavin was therefore not included in the expressioncultures.

Determination of Expression Levels

CYP3A4 content can be determined in whole bacterial cells using Fe²⁺—COvs Fe²⁺ difference spectra, but for the assessment of reductaseexpression levels, bacterial membrane fractions were prepared asdescribed (Gillam et al (1993) Arch. Biochem. Biophys. 305, 123-131).This was necessary because the reductase assay measures the rate ofreduction of cytochrome c, and the background activity measured in JM109cells is prohibitively high to allow direct determination from cells orspheroplasts. For expression of reductase from pB216, the specificactivity of reductase obtained was in the range of 400 pmol reductase/mgmembrane protein, as calculated by rates of reduction of cytochrome c bymembrane preparations. The CYP3A4 content measured in membranes wastypically around 200 pmol/mg membrane protein. Thus after co-expressionof CYP3A4 and reductase from pB216, the bacterial membranes containapproximately a 2:1 ratio of P450 reductase to P450.

Immunodetection of Heterologously Expressed CYP3A4 and Reductase

A typical western blot showing heterologously expressed reductase andCYP3A4 is shown in FIG. 2. Bands corresponding to reductase and CYP3A4can be detected in the membrane fractions derived from pB216 (tracks 5and 6), while reductase alone and CYP3A4 alone can be detected in thosesamples derived from pB207 (tracks 1 and 2) and NF14 (tracks 3 and 4)respectively. Expression was not entirely repressed under non-inducingconditions, as observed from the appearance of reductase and CYP3A4bands in tracks derived from bacterial cultures in which theirexpression had not been induced by the addition of IPTG (tracks 4 and6). However, the amounts of active reductase and spectrally activeCYP3A4 derived from these fractions was found to be much lower thanthose derived from the induced cultures (data not shown), and this mighttherefore indicate that the detection of the bands is not a linearresponse to the content of CYP3A4 and reductase in the samples. Theamounts of heterologously expressed CYP3A4 and reductase in the JM109pB216 membrane fractions appeared similar to those detected in a sampleof human liver microsomes (tracks 6 and 9).

Assays of CYP3A4 Activity in Whole Cells

Whole JM109 pB216 cells were found to metabolise testosterone. No6β-hydroxytestosterone was detected after incubation of testosteronewith the negative control strains JM109 pCW (vector only) or JM109 pB207(reductase expression only). This shows that E. coli is unable tocatalyse 6β-hydroxylation of testosterone in the absence of CYP3A4.Cells expressing CYP3A4 alone (JM109 NF14) also did not producedetectable levels of metabolite, indicating that there is no endogenousprotein in JM109 that can supply electrons to CYP3A4 thereby allowingcatalytic function. When 100 μM cumene hydroperoxide was added to areaction containing JM109 NF14 cells, however, 6β-hydroxytestosteronewas formed. This infers that the CYP3A4 in JM109 NF14 cells isfunctional, but that there is no available intracellular supply ofelectrons. The low activity achieved may reflect an inaccessibility ofthe P450 to cumene hydroperoxide within whole cells. On co-expression ofCYP3A4 with reductase, a relatively high rate of testosterone metabolismoccurs (JM109 pB216 sample). This indicates that co-expressed reductaseand CYP3A4 couple in whole cells to produce a functional monooxygenasesystem.

In this experiment, the 6β-hydroxylase activity was calculated to be˜17.3 nmol 6β-hydroxytestosterone produced/minute/nmol P450. Previousresults with a reconstituted system containing bacterially-expressedCYP3A4 have obtained turnover rates of up to 10 nmol6β-hydroxytestosterone/minute/nmol P450 (Gillam et al (1993) Arch.Biochem. Biophys. 305, 123-131).

Metabolism of Testosterone, Erythromycin and Nifedipine by JM109 pB216Membranes

Membrane fractions derived from pB216 were found to mediate metabolismof the CYP3A4 substrates testosterone, nifedipine and erythromycinwithout supplementation by phospholipids, detergent, glutathione orcytochrome b₅. In these assays, membrane fractions containing 100 pmolCYP3A4 were simply incubated with substrate in the presence of NADPHgenerating system. Typical activities are shown in Table 2. The resultsindicate that JM109 pB216 membrane fractions are proficient formetabolism of three CYP3A4 substrates. The erythromycin N-demethylaseactivity was ˜2.5-fold less than previously observed in reconstitutedsystems containing bacterially-expressed CYP3A4 (Gillam et al (1995)Arch. Biochem. Biophys. 317, 374-384). In contrast, however, thenifedipine oxidase activity was similar to that previously observed fora reconstituted system (Gillam et al (1993) Arch. Biochem. Biophys. 305,123-131).

TABLE 1 Cytochrome P450 content and cytochrome c reductase activities ofJM109 NF14 and 3M109 pB216 cells and/or membranes P450 Content Reductaseactivity nmol/l culture* pmol/mg† nmol cyt c reduced/min/mg pCW n.d.n.d. 38 ± 16 NF14 222 ± 35 350 ± 50 30 ± 3  pJR4 n.d. n.d. 1355 ± 380 pB216 200 ± 45 215 ± 35 1315 ± 321  P450 contents were measured byFe²⁺—CO vs Fe²⁺ difference spectra. Contents are expressed as means of 4experiments ± SD. *Content was measured in 50 μl cells in 1 × TSE (˜0.5ml culture). †Content was assessed per mg protein in membrane fractionsderived from recombinant bacteria. Reductase activities were calculatedby measuring the rate of reduction of cytochrome c per mg protein inmembrane fractions, and values are given as means of four experiments ±SD. n.d. = no detectable activity.

TABLE 2 CYP3A4-dependent metabolism of testosterone, nifedipine anderythromycin by 3M109 pB216 cells and membranes Turnover (min⁻¹)Testosterone Nifedipine Erthro- cells membranes cells membranes mycin*pCW <0.5 <0.5 <2 <2 <0.3 NF14 <0.5 <0.5 <2 <2 <0.3 pJR4 <0.5 <0.5 <2 <2<0.3 pB216 17.3 + 3.3 25.5 + 4.3 15.2 ± 1.3 12.7 ± 0.9 2.3 ± 0.7Metabolism of three known CYP3A4 substrates by cells or membranescontaining recombinant CYP3A4 and P450 reductase was assessed. Turnovernumbers are recorded as nmol product formed/min/nmol P450, and are shown± SD. The products which were detected were 6β hydroxytestosterone,oxidised nifedipine and formaldehyde. Where no activities were detected,detection levels are shown. For testosterone metabolism, no 6βhydroxytestosterone was formed even after 60 minutes incubation # withcells or membranes lacking either CYP3A4 or P450 reductase (data notshown). *For erythromycin metabolism, activities could only be recordedwith membranes, as the background level of formaldehyde formation bywhole cells was very high.

Discussion

In this Example we describe the generation of a functional P450monooxygenase system in E. coli, by co-expression of the cDNAs encodinghuman CYP3A4 and P450 reductase. To our knowledge, this is the firstinstance in which a mammalian xenobiotic-metabolising P450 has beenshown to be catalytically active in intact E. coli cells. While thesteroidogenic P450 17α-hydroxylase/17-, 20-lyase (P450c17) is functionalin E. coli, by virtue of its ability to accept electrons from thebacterial flavodoxin/NADPH-flavodoxin reductase system (Jenkins et al(1994) J. Biol. Chem. 269, 27401-27408), E. coli cells expressing CYP3A4alone (JM109 NF14 cells) did not metabolise the CYP3A4 substratetestosterone in the absence of an exogenous supply of electrons (in thiscase from cumene hydroperoxide). We therefore conclude that CYP3A4cannot couple with this or another bacterial reductase.

We developed a system for co-expression of CYP3A4 and human P450reductase in E. coli, in order that these cells might be of use asbiocatalysts for production of valuable P450 metabolites. Both cDNAswere expressed under separate P_(tac)P_(tac) promoters in a singleplasmid, pB216, so that coordinate expression of the CYP3A4 andreductase could be induced by IPTG. To achieve optimal levels ofexpression, both cDNAs were modified from their original form. The 5′end of the CYP3A4 cDNA was modified as previously described (Gillam etal (1993) Arch. Biochem. Biophys. 305, 123-131). For efficientexpression of P450 reductase, we found that it was necessary to extendthe 5′ end of the cDNA by fusion with the sequence encoding thebacterial pelB signal peptide. Yields of CYP3A4 of 200 pmol/mg membraneprotein, and reductase yields 400 pmol/mg membrane protein wereobtained.

We believe that the co-expressed CYP3A4 and pelB-reductase may belocalised on the same face of the bacterial cytoplasmic membrane, asJM109 pB216 cells and membranes isolated from them are active towardsCYP3A4 substrates, indicating that the proteins must be able to coupleefficiently. Activities measured towards the substrates were found to behigher than previous results obtained with purifiedbacterially-expressed CYP3A4 in a reconstituted system (Gillam et al(1993) Arch. Biochem. Biophys. 305, 123-131; Gillam et al (1995) Arch.Biochem. Biophys. 317, 374-384). Such reconstituted systems containpurified CYP3A4, reductase, cytochrome b₅, glutathione, detergent and anoptimised phospholipid composition. Cytochrome b₅ has been shown to beimportant in obtaining maximal CYP3A4 activities against particularsubstrates for a CYP3A4-reductase fusion protein (Shet et al (1993)Proc. Natl. Acad. Sci. USA 90, 11748-11752), while the phospholipidcomposition and glutathione concentration was also shown to be criticalin a reconstituted system containing CYP3A4 (Gillam et al (1993) Arch.Biochem. Biophys. 305, 123-131). In the absence of these ancillaryfactors, CYP3A4 activity towards testosterone was rather unexpected inwhole cells and in membranes within the simple buffer system used here.It was therefore extremely interesting that, in our studies, high levelsof CYP3A4 activity towards these substrates was observed in the absenceof exogenously added cytochrome b₅.

In summary, we have successfully achieved high level co-expression ofCYP3A4 and human P450 reductase in E. coli. The resulting strain isproficient for whole cell metabolism of testosterone and nifedipine evenin the absence of exogenously applied NADPH, suggesting that (at least)the reductase active site is cytoplasmically orientated. Membranesderived from the bacteria metabolise testosterone, nifedipine anderythromycin in a simple buffer supplemented only with NADPH. Thespecific activities achieved from our co-expression strain are higherthan previously obtained with reconstituted systems. We hope toinvestigate the possible reasons for this discrepancy to improve theturnover rates. The strain described in the present Example will be ofuse as a biotechnological tool for production of CYP3A4 metabolites, andwill be used as a model system for future co-expression of alternativeP450 isoforms with P450 reductase in E. coli.

EXAMPLE 2

Materials and Methods

Bacterial Strains and Plasmids

The E. coli K-12 strain JM109 (Yanisch et al (1985) Gene 33, 103-19) wasused throughout. Cytochrome P450 cDNAs were expressed from the plasmidpCW. This plasmid includes a β-lactamase gene, and can therefore bestably maintained in bacterial cells grown in the presence of an agentsuch as ampicillin or carbenicillin. The human P450 reductase cDNA wasexpressed from the plasmid pACYC184, which can be stably maintained inbacterial cells in the presence of chloramphenicol.

Isolation of Bacterial Signal Peptide Coding Sequences and Generation ofExpression Constructs

The source of the coding sequence for the bacterial pelB signal peptidewas the commercial vector pET-20b (Novagen). Chromosomal DNA wasextracted from E. coli strain JM109 and used as the template for theisolation of the ompA leader sequence by PCR, using specificoligonucleotide primers. This PCR product was sub-cloned and subjectedto dideoxy sequencing, and was found to be identical to an ompA leadersequence already registered in the GenEMBL database (accession number :v00307), namely:5′ATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCC-3′ (SEQ ID No 6)

A PCR fusion technique (Yon et al (1989) Nucleic Acids Res. 17, 4895)was used to fuse the coding sequence for the bacterial pelB or ompAsignal peptide in frame to the 5′-end of the full length P450 cDNA.Using this method, we made pelB-CYP3A4, ompA-CYP3A4, ompA-CYP2D6,ompA-CYP2A6, and ompA-CYP2E1 fusions, engineering in each case a NdeIrestriction site at the 5′ end of the construct to facilitatesub-cloning into pCW. All PCR-derived fragments were verified by dideoxysequencing.

The pelB-human reductase construct described previously was sub-cloned,together with an upstream (tac)₂ promoter cassette, as a BclI-BglIIfragment into the unique BamHI site of the plasmid pACYC184, to produceplasmid pJR7.

Expression in E. coli of Human P450s Fused to Bacterial Leader Sequences

JM109 cells were transformed with pCW/pelB-CYP3A4, pCW/ompA-CYP3A4,pCW/ompA-CYP2D6, pCW/ompA-CYP2A6, or pCW/ompA-CYP2E1 (Inoue et al (1990)Gene 96, 23-28). Transformed cells were selected with ampicillin andamplified for further study. For co-expression, JM109 cells wereco-transformed with the plasmids pCW/ompA-CYP2D6 and pJR7, and selectedon both ampicillin and chloramphenicol.

A standard protocol was used for all expression and co-expressionexperiments. Transformants were streaked from a frozen glycerol stockonto LB agar plates containing either ampicillin alone (for P450expression) or ampicillin plus chloramphenicol (for co-expression ofP450 and reductase), and incubated at 37° C. for 12-14 h. Singleisolated colonies were then inoculated into a few ml of LB broth(containing the appropriate antibiotic(s)) and shaken overnight at 37°C. This starter culture was diluted 1:100 into 100 ml Terrific Brothmodified and supplemented as described (Gillam et al (1993) Arch.Biochem. Biophys. 305, 123-131), but with chloramphenicol (25 μg/ml)added when reductase was being co-expressed, in a 1 l conical flask.These cultures were incubated at 30° C. for 4-5 h with shaking. The haemprecursor δ-aminolevulinic acid was then added to a final concentrationof 0.5 mM, and the inducing agent IPTG to 1 mM. Expression ofheterologous proteins was then allowed to proceed for 22-23 h at 30° C.

Harvesting Cultures and Determination of Expression Levels

as described previously

Immunodetection of Heterologously Expressed P450 and Reductase

Proteins were separated on SDS-9% acrylamide gels and then transferredonto Hybond-ECL membrane (Amersham, UK). The membrane was blocked with5% milk powder in TBS-X [50 mM Tris. Cl (pH 7.9), 150 mM NaCl, 0.10%Triton X-100] for 1 h, then incubated with diluted primary antibodiesfor 45-60 min. The primary antibodies used were rabbit anti-CYP3A,rabbit anti-CYP2D6, rabbit anti-reductase, or sheep anti-CYP2E1immunoglobulins. The membrane was washed in four changes of TBS-X, andthen incubated with the secondary antibody (HRP-linked donkey,anti-rabbit or anti-sheep IgG, as appropriate) for 25-35 min. After fourwashes with TBS-X, the recombinant proteins were detected bychemiluminescence using ECL (Amersham, UK). Secondary antibodies wereobtained from the Scottish Antibody Production Unit.

Bufuralol 1′-hydroxylase Assays

Assays on bacterial membrane fractions were carried out at 37° C. in 50mM potassium phosphate buffer, pH 7.4, containing 20 or 50 pmol CYP2D6,50 μM (±)-bufuralol, and a NADPH regenerating system (see Example 1) ina total volume of 300 μl. For membranes isolated from cells carryingpCW/ompA-CYP2D6 only (ie. no reductase co-expressed), the NADPHregenerating system was replaced by 100 μM cumene hydroperoxide.Reactions were stopped by the addition of 15 μl of 60% perchloric acidand placed on ice for 5-10 min. Precipitated proteins were removed bycentrifugation, and then the supernatants were analysed for1′-hydroxybufuralol by reversed-phase HPLC, with reference to authenticstandard. Separation was achieved using a Spherisorb 5 μm ODS-2 column25 cm×4.6 mm, and a mobile phase of 0.1 M ammonium acetate (pH 5.0) witha linear gradient of acetonitrile (27 to 51% in 12 min). Detection wasby fluorescence using excitation and emission wavelengths of 252 and 302nm, respectively.

Assays on whole cells were carried out at 37° C. in 50 ml conicalflasks, and contained 50 pmol CYP2D6, 50 μM (±)-bufuralol and 1×TSEbuffer in a total volume of 5 ml. Samples (300 μl) were withdrawn at 0,2, 5 and 10 min into tubes containing 15 μl of 60% perchloric acid onice. Analysis then proceeded in an identical manner to the membraneassays (see above).

For the determination of Michaelis-Menten kinetic parameters,incubations were carried out for 5 min with 20 pmol CYP2D6 and varyingconcentrations of substrate (0 to 100 μM). K_(m) and v_(max) wereestimated from double-reciprocal plots of initial reaction velocityversus substrate concentration.

Results

Isolation of P450 and P450-reductase cDNAs

The cDNAs for CYP3A4, CYP2D6, CYP2A6 and CYP2E1 and P450-reductase wereisolated by RT-PCR using amplimers which were synthesized based on thepublished sequence information. The cDNAs were verified byDNA-sequencing and found to encode proteins with primary structuresidentical to those published for CYP3A4, CYP2D6, CYP2A6 and CYP2E1 andthe human P450-reductase.

Construction and Expression of Human P450 Reductase Fused to the pelBLeader Sequence

P450 reductase is required for the catalytic activity of human P450s andis absent in E. coli. We tried to express the native P450 reductase orthe P450 reductase fused at its N-terminus to the pelB leader sequence(MKYLLPTAAAGLLLLAAQPAMA-) (SEQ ID No 1) in E. coli. Initially we triedto express both proteins under the control of the IPTG(isopropylthiogalactoside) inducible (tac)₂ promoter in the plasmid pCW(FIG. 3). Membranes were isolated from E. coli harbouring the nativeP450-reductase and the pelB-reductase and were analysed byimmunoblotting (FIG. 4). In the absence of IPTG, negligible amounts ofP450 reductase were detected, and a clear induction of the recombinantprotein was observed upon addition of IPTG. Expression of the nativeP450 reductase from the plasmid pJR1 was low, whereas high levels ofpelB-P450 reductase were achieved from the plasmid pJR2. Thesedifferences were also reflected in the P450-reductase activity towardscytochrome c detected in membranes containing these two recombinantproteins. In these preparations the pelB-reductase displayed an almostten fold higher reductase activity as compared to the native P450reductase (FIG. 4). These results clearly demonstrate that optimalexpression of functional P450 reductase in E. coli is only possibleafter fusion of this protein to a bacterial leader sequence.

Construction and Expression of CYP3A4 Fused to Bacterial LeaderSequences

CYP3A4 is the major human hepatic P450 (Shimada et al (1994) J.Pharmacol. Exp. Ther. 270, 414-423) and is involved in the metabolism ofa wide variety of therapeutic drugs as well as chemical carcinogens. Inorder to achieve efficient expression of this important P450, weconstructed a series of modified CYP3A4 cDNAs which encode the CYP3A4fused at its N-terminus to either the bacterial pelB or the bacterialompA leader sequence (MKKTAIAIAVALAGFATVAQA) (SEQ ID No 2) using a PCRbased strategy which has been shown to allow the fusion of any twosequences at any site (Yon et al (1989) Nucleic Acids. Res. 17, 4895).The resulting cDNA constructs were sequenced, cloned into the vector pCWand were transformed into the E. coli strain JM109 for expression.

Colonies which grew in the presence of ampicillin were selected forfurther DNA and protein analysis. E. coli harbouring the expressionconstructs were grown in terrific broth and P450 expression was inducedby IPTG as described in Materials and Methods. CYP3A4 expression wasdetected by immunoblot analysis of bacterial membranes (FIG. 5) and byspectral analysis of whole cells or membranes (FIG. 6). As can be seenfrom FIG. 5, the level of immunoreactive pelB-CYP3A4 was similar to theCYP3A4 level found in human liver microsomes, whereas the level ofimmunoreactive ompA-CYP3A4 was at least an order of magnitude higher.Interestingly, pelB-CYP3A4 yielded two closely migrating immunoreactiveproteins. A similar result was also obtained after purification of aHis-tagged pelB-3A4 by nickel-agarose affinity chromatography, whereasompA-3A4 which had been purified similarly yielded a homogeneousprotein. pelB-CYP3A4 and ompA-3A4 displayed a Fe²⁺ vs. Fe²⁺ —CO spectrumtypical for P450 haemoproteins (FIG. 6). The yield of spectrally activepelB-CYP3A4 but not of ompA-CYP3A4 protein was strongly stimulated bythe presence of δ-aminolaevulinic acid (ALA) in the growth medium (FIG.7). Under these conditions ompA-3A4 yielded more spectrally active P450compared to peIB-CYP3A4 (500 nmoles/l culture vs. 143 nmole/l culturerespectively) even though the difference was less pronounced thanexpected from the immunoblot analysis. The presence of ALA in theculture medium led to the appearance of an absorption peak at 420 nm inthe spectral analysis of the recombinant P450s (see FIG. 6, pelB-3A4expressed in the presence of ALA and ompA-3A4 expressed in the absenceof ALA).

Direct determination of the catalytic activity of the expressed proteinswas not possible due to the absence of P450 reductase in bacterialmembranes. The P450 enzyme activity was therefore determined in thepresence of cumene hydroperoxide which serves as an artificial oxygendonor for P450s. In this analysis, we found that the turnover number ofthe pelB-CYP3A4 and the ompA-3A4 for the 6β-hydroxylation oftestosterone was 4.2 min⁻¹ and 3.2 min⁻¹ respectively. These resultsclearly demonstrate that in E. coli these P450s fold correctly intospectrally and catalytically active enzymes.

Construction and Expression of CYP2D6 Fused to the ompA Leader Sequence

As we had found that the yield obtained for ompA-CYP3A4 was much higherthan for the pelB-CYP3A4, we decided to fuse exclusively the ompAsequence to the N-terminus of CYP2D6, which is a P450 involved in themetabolism of a variety of therapeutically important compounds.

The PCR technique used for the fusion of the ompA signal sequence to theCYP2D6 cDNA was similar to the strategy used for the construction of theompA-CYP3A4. This construct was expressed from the vector pCW. FIG. 8displays an immunoblot of bacterial membranes obtained from E. coliharbouring the ompA-CYP2D6 cDNA construct (lane 2). An immunoreactiveband corresponding to recombinant CYP2D6 was detected in thesemembranes, which was absent in membranes isolated from bacteria carryingthe empty expression plasmid, pCW (lane 1). The ompA-CYP2D6 yielded atypical P450 Fe²⁺ vs. Fe²⁺ —CO spectrum (FIG. 9). The yield ofspectrally active CYP2D6 (481 nmoles/l of culture, FIG. 10) was similarto the yield achieved for ompA-CYP3A4 and resulted in a distinct redcolouration of the bacteria expressing the former hemoprotein. The ALAdependent increase of spectrally active ompA-CYP2D6 was much strongerthan for pelB-CYP3A4. In the presence of cumene hydroperoxide,ompA-CYP2D6 catalyzed the hydroxylation of the typical CYP2D6 substratebufuralol with a turnover number of 50±3 min⁻¹.

Co-expression of P450 and P450-reductase Fused to Bacterial LeaderSequences Generates a Functional Monooxygenase System in E. coli

In order to generate a functional P450-monooxygenase system, we tried toco-express P450 and P450-reductase in E. coli. It is possible toenvisage three main ways to achieve co-expression of two proteins in E.coli. Firstly, two cDNAs can be expressed from separate compatibleplasmids in the same cell: for example, P450 could be expressed from pCWand P450 reductase from a separate vector. Secondly, both cDNAs could besubcloned into the same plasmid. Thirdly, one or both of the cDNAsencoding P450 or P450 reductase could be integrated into the bacterialchromosome. We chose the first strategy, as it is technically leastdemanding.

The pelB-reductase cDNA was cloned into the vector pACYC184 (FIG. 3) toyield the vector pJR7. The advantage of this construct is that pACYC184contains a different origin of replication from pCW. In addition thesevectors contain different selection markers, which allow stablecotransformation with two separate plasmids, one for the expression ofP450-reductase (pACYC 184), the other (pCW) for expression of P450. E.coli were transformed with pJR7 and pCW carrying the ompA-CYP2D6 cDNA.Transformants were selected on ampicillin and chloramphenicol andexpanded for further analysis. Immunoblotting (FIG. 8) revealedimmunoreactive bands corresponding to the recombinant P450 reductase andCYP2D6 in membranes isolated from the co-expressing strains (lanes 3 and4), which were both absent in membranes isolated from bacteria carryingthe empty expression vector, pCW (lane 1). E. coli co-expressingompA-CYP2D6 and the pelB-reductase displayed a typical Fe²⁺ vs. Fe²⁺ —COspectrum (FIG. 9). The total cellular yield of spectrally activeompA-CYP2D6 was 365 nmoles/l culture in the co-expressing strain whichwas only 25% lower than in E. coli expressing CYP2D6 alone (FIG. 10).Membranes containing the ompA-CYP2D6 and P450-reductase displayed acytochrome c reductase activity of 530 nmoles/min/mg which is two foldhigher than the value reported for human liver microsomes. Mostimportantly intact bacteria co-expressing ompA-CYP2D6 and pelB-reductaseas well as membranes derived from them catalyzed the hydroxylation ofthe typical CYP2D6 substrate bufuralol extremely efficiently withturnover numbers of 4.6 min⁻¹ and 5.7 min⁻¹ respectively and a specificactivity (1.2 nmoles/min/mg membrane protein) which was 40 fold higherthan the value reported for human liver microsomes. The V_(max) and theK_(m) for the CYP2D6-catalyzed bufuralol 1′-hydroxylation were found tobe 13.3 min⁻¹ and 11.1 μM respectively (FIG. 11).

Construction and Expression of CYP2A6 Fused to the OmpA Signal Peptide

The ompA signal peptide was fused to the N-terminus of full-lengthCYP2A6 by PCR. The recombinant ompA-CYP2A6 expressed from pCW displayeda typical Fe²⁺ vs. Fe²⁺—CO difference spectrum in both whole cells andmembranes derived from these cells (FIG. 13). The yield of spectrallyactive CYP2A6 in whole cells (193 nmol/l culture, FIG. 14) was somewhatlower than that of either CYP3A4 or CYP2D6 expressed from the analogousconstruct. Nonetheless, the specific CYP2A6 content of membranesisolated from these cells greatly exceeded the level which would befound in human liver microsomes.

Construction and Expression of CYP2E1 Fused to the OmpA Signal Peptide

The same PCR-based strategy was used to fuse the ompA signal peptide tothe N-terminus of full-length human CYP2E1. Expression from pCW resultedin the appearance of a protein in bacterial membranes which wasdetectable on immunoblotting using specific anti-CYP2E1 antibodies (FIG.15, lane 3), and which was of the same apparent size as CYP2E1 in asample of human liver microsomes (FIG. 15, line 1). This protein wasabsent in a sample of membranes isolated from E. coli carrying the emptyexpression plasmid, pCW (lane 2). The relative band intensities (cf.lanes 1 and 3) suggest that a very high level of recombinant CYP2E1 hasbeen produced in this bacterial strain.

Furthermore, expression from pCW/ompA-CYP2E1 produced a typical reducedFe-CO difference spectrum in whole bacterial cells (FIG. 16), althoughin this instance, the absorption maximum at around 420 nm was rathermore pronounced than for the other P450-expressing constructs. The yieldof spectrally active CYP2E1 in whole E. coli carrying pCW/ompA-CYP2E1was estimated to be 451 nmol/l culture—approximately the same as thelevels of CYP3A4 and CYP2D6 produced in whole cells from the analogousompA-constructs.

Discussion

This Example demonstrates that a highly functional monooxygenase systemin E. coli can be made by the fusion of P450s as well as P450 reductaseto bacterial leader sequences such as pelB and ompA. This is clearlyillustrated by the almost ten fold increase of functional P450 reductaselevels obtained after fusion of the native P450 reductase to the pelBleader sequence and the expression of this construct from the vector pCW(FIG. 1). Expression of pelB-reductase from the vector pACYC 184, whichwe employed for the co-expression of P450s and P450-reductase, yieldedP450-reductase levels in E. coli membranes similar to those found inhuman liver microsomes (100 pmol P450-reductase/mg protein). Theapplicability of our approach is not only limited to the expression ofP450-reductase in E. coli, since we found that pelB-P450 reductase canalso be expressed in S. typhimurium at levels similar to those achievedin E. coli (data not shown).

Moreover, we demonstrate that P450s fused to bacterial leader sequencescan be efficiently expressed in E. coli without relying on the extensivemodifications of P450 N-termini which previously had been thought to beessential for optimal P450 expression (Barnes et al (1991) Proc. Natl.Acad. Sci. USA 88, 5597-5601; Gillam et al (1993) Arch. Biochem.Biophys. 305, 123-131; Larson et al (1991) J. Biol. Chem. 266,7321-7324). Our gene fusion approach is applicable for P450s belongingto different gene families, since we were able to show that CYP3A4,CYP2D6, CYP2A6 and CYP2E1 (FIGS. 5, 8 and 15) can all be expressed in E.coli using this strategy. Recently we have also extended this approachto the expression of CYP2C9 and CYP2D9.

Previously CYP3A4 and CYP2D6 cDNAs have been expressed in E. coli aftermodifications of their 5′ ends in order to remove regions with thepotential to form secondary structures (Gonzalez et al (1995) Annu. Rev.Pharmacol. Toxicol. 35, 369-390). These modifications translated intoextensive changes in the N-terminal region of CYP3A4 and CYP2D6 (in thefollowing these modified proteins are designated as 17α-CYP3A4 and17α-CYP2D6 respectively). With respect to P450 yield our gene fusionstrategy is superior to this approach, at least for the P450s which wehave tried, since yields of ompA-CYP3A4 and of ompA-CYP2D6 expressedfrom the vector pCW were at least by a factor of 1.7 and 4.8respectively higher than the published values for 17α-CYP3A4 and of17α-CYP2D6 expressed from the same vector (FIG. 12). We strengthenedthis observation by repeating the expression of 17α-CYP3A4 and17α-CYP2D6 from constructs which had been generated in our laboratoryaccording to published procedures (Gillam et al (1993) Arch. Biochem.Biophys. 305, 123-131; Gillam et al (1995) Arch. Biochem. Biophys. 319,540-550). We have expressed the pelB-CYP3A4 and the ompA-CYP3A4 withC-terminal extensions containing a stretch of histidine residues. Wewere able to purify mg quantities of these proteins for structuralanalysis using nickel agarose affinity chromatography.

We have extended our ompA leader fusion strategy to allow the expressionof two further human cytochromes P450, namely CYP2A6 and CYP2E1, in E.coli. For CYP2E1, our expression level of 451 nmol/l culture (FIG. 16)is an order of magnitude greater than the previously published level ofexpression for this enzyme in E. coli (40 nmol/l, Gillam et al (1994)Arch Biochem Biophys 312, 59-66), using conventional sequenceoptimisation procedures. To our knowledge, the expression of CYP2A6 inE. coli has never before been reported. Hence, we have been able todemonstrate the simplicity and versatility of our system.

Most importantly we show that the gene fusion approach can be used togenerate a highly functional human P450 monooxygenase system in E. coli.This is clearly demonstrated by the high bufuralol 1′-hydroxylaseactivity observed with intact E. coli co-expressing ompA-CYP2D6 andpelB-P450 reductase. This activity (1.2 nmoles/min/mg protein) is about20 fold higher than the average bufuralol hydroxylase activity reportedfor a panel of human liver microsomes. Also bacterial membranes isolatedfrom this E. coli strain display a similar high P450 enzyme activity.The ompA-CYP2D6 displays a higher substrate turnover number (4.6 min⁻¹)than the value calculated for human liver microsomes (1 min⁻¹) and alsothan the data reported for CYP2D6 reconstituted in an optimized cellfree system. This result clearly demonstrates that ompA-CYP2D6 and thepelB-reductase couple very efficiently in E. coli and suggests that bothproteins are located on the same side of the bacterial membrane in aphospholipid environment at least as optimal as the complex phospholipidcompositions employed in reconstituted systems.

It is important to note that E. coli expressing ompA-CYP2D6 andpelB-reductase use endogenous NADPH as source for reducing equivalents,since they displayed P450 enzyme activity in the absence of exogenouslyadded NADPH. This finding suggests that the reductase active site islocated on the cytoplasmic side of the inner membrane where it is ableto utilise the intracellular pool of NADPH. This property, combined withthe high yield of recombinant protein and the technically simplemaintenance, makes the system ideally suited for bioreactor purposes.However we have observed that P450 substrates are metabolized poorly byE. coli co-expressing P450s and P450 reductase when maintained inculture broth rather in the buffer used in the present study. This mightindicate that under the former conditions, substrates were not able topenetrate the bacterial cell wall and membranes. We have therefore takenour strategy further and have expressed a functional P450 monooxygenasesystem in the TA series of S. typhimurium strains. These strains havefrequently been used for mutagenicity testing, since they contain a deeprough mutation resulting in a permeable cell wall, which can bepenetrated by a large panel of structurally diverse compounds (Ames etal (1975) Mutat. Res. 31, 347-364; Simula et al (1993) Carcinogeizesis14, 1371-1376). We were able to show that using our gene fusion strategya functional P450-monooxygenase system could be generated in S.typhimurium.

We envisage that the P450 and P450-reductase expression levels in E.coli can be further increased using other vectors or other bacterialhosts. For example we have observed that some of the bacteriallyexpressed ompA-CYP2D6 was malfolded and might have formed inclusionbodies. Recently E. coli strains have become available which expressmolecular chaperones and thioredoxin (Yasukawa et al (1995) J. Biol.Chem. 270, 25328-25331). The expression of these proteins, which preventmisfolding of newly synthesized proteins, resulted in a much higheryield for several recombinant proteins. These strains could be also usedfor the expression of P450s from expression vectors containing strongerbacterial promoters than pCW. For example we have observed that highlevels of recombinant CYP3A4 can be expressed in E. coli under thecontrol of the powerful T7 polymerase promoter in the pET series ofvectors. However only a small fraction of the recombinant CYP3A4 wasspectrally active. Furthermore we envisage that the coupling of P450swith P450-reductase can be further improved by co-expression of theP450-reductase FMN domain which has been recently shown to stimulate theP450 enzyme activity in a reconstituted system containing P450 and P450reductase.

In summary, we have developed a general approach for the efficientbacterial expression of P450s and demonstrated that this approach can beused to generate bacteria containing a highly functional P450 dependentmonooxygenase system. These models will have important commercialapplications in drug development and biocatalysis.

EXAMPLE 3

Expression in Salmonella typhimurium

Coexpression of P450 and P450s in certain S. typhimurium strains such asTA1538 and TA1535 is readily adapted from the E. coli system describedabove. The same vector system can be employed in both species. Thevector is first passaged from the E. coli strains which are used forP450 expression (eg. JM109 or DH5α) through the E. coli strain LA5000and from there to the S. typhimurium strains. However for the expressionof P450s in the S. typhimurium strains TA98 and TA100, which arefrequently used in mutagenicity testing, the strategy may have to beslightly modified. TA98 and TA100 carry the plasmid pKM101 whichincreases the SOS repair in these strains and concomitantly theirsensitivity to mutagens. However this plasmid codes also for theampicillinase. For the expression of P450s from the expression plasmidpCW in these strains, the ampicillinase marker on pCW has to be replacedby a tetracycline resistance marker. Otherwise growth conditions andinduction of P450 expression in S. typhimurium is similar to the E. colisystem. However, we have found that it is desirable to omit tetracyclinefrom the growth medium during the P450 induction phase.

EXAMPLE 4

Expression of CYP3A4 and P450 Reductase in Escherichia coli andSalmonella typhimurium Strains with Altered Outer Membrane Permeability

Materials and Methods

Bacterial Strains and Plasmids

The E. coli K12 strains used were JM109 (Yanisch et al (1985) Gene 33,103-19), AB1157 (Howard-Flanders et al (1964) Genetics 49, 237-246) andNS3878 (Chaterjee and Sternberg (1995) Proc. Natl. Acad. Sci. 92,8950-8954). The S. typhimurium strain was TA1535 (Ames et al (1975)Mutat. Res. 31, 347-352)). NS3678 is strain AB1157 tolC (λLP1) and thetolC mutation is due to a Tn10tet^(r) insertion. TolC⁻ mutants areextremely sensitive to hydrophobic agents (Whitney (1970) Genetics 30,39-59) and this protein is proposed to play a role in assembly oflipopolysaccharide (LPS) in the outer membrane (Schnaitmann and Klena(1993) Microbiol. Rev 57, 655,682). S. typhimurium strain TA1535 carriesan rfa mutation and has a defective outer membrane. Co-expression ofCYP3A4 and P450 reductase was achieved using the plasmid pB216 describedin the previous examples.

Co-expression of CYP3A4 and P450 Reductase

The protocol used for expression was identical to those previouslydescribed with the exception that for NS3678 [pB216] tetracycline(10μg/l) was included in the streak plates and in the overnight LBculture but not in the terrific broth. CYP3A4 content was determined inwhole bacterial cells using Fe²⁺—CO vs Fe²— difference spectra.

Incubation of Intact Cells with CYP3A4 Substrates

After the usual 20-24 hr induction, cells were cooled on ice for 10minutes before harvesting by centrifugation. The cells were then washedonce in an equal volume of ice cold 1×M9 salts solution beforeresuspending in {fraction (1/10)} volume of M9+glucose (10 mM). Care wastaken throughout not to subject the cells to vigorous pipetting. Cellsuspensions were stored on ice until required for incubation with CYP3A4substrates, 500 μl was removed and added to a 50 ml polypropylene tubecontaining 4.5 ml M9 +glucose. The tubes were pre-incubated for 5-10minutes in an orbital shaker at 37° C. before the reactions wereinitiated by addition of either testosterone or nifedipine at a finalconcentration of 200 μM. When required, 200 μl of cell suspension wasremoved and transferred to a 1.5 ml Eppendorf microcentrifuge tubecontaining 100 μl of methanol and 5 μl of 60% perchloric acid. The tubeswere mixed by inversion and stored on ice for 10 minutes beforecentrifuging to remove precipitate. The supernatants were transferred tomicrovials for HPLC analysis. Metabolites were separated as previouslydescribed and the yield of 6β-OHT) or nifedipine oxide calculated byreference to known standards.

Results

CYP3A4 and P450 Reductase Expression in the Four Strains

Levels of P450 expression and P450 reductase activity are shown in TableA. CYP3A4 content in NS3678 [pB216] cells is significantly lower than inthe other three strains, typically around 30 nmol/l. The reasons forthis are unclear and expression in this strain will require furtheroptimisation.

TABLE A Expression levels of CYP3A4 in and CYP3A4-dependent metabolismof testosterone and nifedipine by intact JM109, AB1157, NS3678 andTA1535 pB216 cells P450 content (nmol/l Turnover (min⁻¹) culture)Testosterone Nifedipine JM109 [pB216] 105 ± 13 0.15 ± 0.11 1.38 ± 0.06AB1157 [pB216]  80 ± 24 2.5 18.4 NS3678 [pB216] 32 ± 7 15.3 ± 12.3 17.8± 2.9  TA1535 [pB216] 119 ± 42 0.52 ± 0.14 5.4 ± 2.5 P450 contents weremeasured by Fe²⁺—CO vs Fe²⁺ difference spectra. Contents are expressedas means of at least 3 experiments ± SD. Turnover numbers are recordedas nmol product formed/min/nmol P450, and are shown ± SD. The productsdetected were hydroxytestosterone and nifedipine oxide.

Metabolism of Testosterone and Nifedipine by intact E. coli and S.typhimurium Strains Expressing CYP3A4 and P450 Reductase

The turnovers for testosterone and nifedipine by the four strains areshown in Table A. From previous work using JM109 [pB216] it had beenshown that metabolism of testosterone was negligible unless the cellswere resuspended in a buffer (TSE) containing EDTA and subsequentlysubjected to osmotic shock. This was confirmed here, the turnover aftera 10 minute incubation being only 0.15 nmol 6β-OHT/nmol P450/min. Incontrast testosterone metabolism in the other two E. coli strains weremuch more extensive, NS3678 [pB216] being particularly impressive with aturnover of 15.3 which is 100-fold higher than in JM109 [pB216]. The S.typhimurium strain TA1535 [pB216] also showed more activity than JM109[pB216] towards testosterone but only by a factor of 3-4 fold. Theturnovers for nifedipine followed a similar pattern although theinter-strain differences were not so marked as with testosterone. The E.coli strains NS3678 [pB216] and AB1157 [pB216] again showed the highestactivity at around 18 nmol nifedipine oxide/nmol P450/min with turnoversfor TA1535 [pB216] and JM109 [pB216] being around 3 and 15-fold lowerrespectively.

The results of time course incubations with the two substratesnifedipine and testosterone are shown in FIGS. 17 and 18 respectively.In the two most extensively metabolizing strains, AB1157 and NS3678,metabolite accumulation continues for 1-2 hours and with finalconcentrations of metabolite in the 30-40 μM range representing a yieldof 20-30%. The loss of linearity after approximately one hour incubationmay be due to loss of CYP3A4 or metabolite inhibition.

Discussion

Work described in the previous examples shows that JM109, the strain ofE. coli routinely used for expression, was unable to catalysetestosterone hyroxylation when co-expressing CYP3A4 and reductase unlessthe cells were subjected to osmotic shock in TSE buffer. This lack ofmetabolism is almost certainly due to the impermeability of the E. coliouter membrane to large, hydrophobic molecules (Nikaido and Vaara (1985)Microbiol. Rev 49, 1-32) and makes this strain unsuitable for generationof large quantities of certain P450 metabolites in bioreactor systems.It is anticipated that outer membrane permeability will not be a problemfor all P450 substrates, for instance we have found that turnover of7-ethoxyresorufin in intact JM109 expressing CYP1A2 and P450 reductaseis comparable to that in osmotically shocked cells (data not shown). E.coli strains such as NS3678 which carry a mutant tolC gene are known tobe hypersensitive to hydrophobic agents and it was predicted thatpermeability to P450 substrates would also be increased. Based on theextremely high turnovers of testosterone and nifedipine by NS3678[pB216] relative to JM109 [pB216] this seems to be the case. Currently,the P450 expression levels in strain NS3678 are relatively low. Weenvisage that these can be improved by optimisation of growthconditions. However, expression levels in the parent strain, AB1157[pB216], are comparable to those in JM109 [pB216] with the advantagethat substrate turnover is considerably higher. We believe that this isdue to the rfbD1 mutation carried by this strain which confers apartially defective outer membrane. Our work represents the firstexample of expression of a functional P450 monooxygenase in “permeable”strains of E. coli. The system described here will be applicable forexpression of all P450s and will facilitate the production of a widerange of P450 metabolites in “bioreactor” systems.

Summary

E. coli JM109 or DH5α which coexpressed P450s together with P450reductase metabolised substrates only after treatment with buffer (TSE)which would alter the permeability of membranes. We have now expressedthese enzymes in S. typhimunum TA1535 and in E. coli strains with apermeable cell wall. We were able to shown that, in the presence of aphysiological broth (minimal medium+glucose), the metabolism ofsubstrates is linear for more than 60 minutes at turnover rates whichapproach those found using cells resuspended in TSE buffer.

EXAMPLE 5

Co-expression of OmpA- and OmpA (+2)-P450s with Reductase

Materials and Methods

Plasmids

Two P450 expression plasmids, pCW/ompA(+2)-CYP3A4 andpCW/ompA(+2)-CYP2A6, were constructed. In each case, PCR was used toinsert a dipeptide “linker” (-Ala-Pro-), corresponding to the first twoamino-acids of the mature OmpA protein, between the OmpA signal peptideand the P450 N-terminus. In order to allow facile purification ofrecombinant CYP3A4 by affinity chromatography (see below) for N-terminalsequence analysis, two further plasmids, pCW/ompA-CYP3A4(His)₆, andpCW/ompA(+2)-CYP3A4(his)₆ were also constructed, in which six histidineresidues were appended to the C-terminus of the P450.

Co-expression Methodology

The basic techniques for co-expression of P450 and reductase fromseparate compatible plasmids have already been described in the previousexamples.

Coumarin 7-hydroxylate Assay

membrane assays were carried out in 100 mM Tris-HCl (pH 7.4) containing20 pmol CYP2A6, 50 μM coumarin, and a NADPH generating system (describedpreviously in Example 1), in a total volume of 500 μl. Assays onbacterial cells were performed in TSE buffer in a total volume of 5 ml,and contained 50 pmol CYP2A6 per ml incubation, and 50 μM coumarin.Samples (500 μl) were withdrawn at intervals and analysed for metaboliteformation. Processing of samples from cell and membrane assays wasidentical: reactions were stopped by the addition of 72 μl of 12.5%trichloroacetic acid, and were then placed on ice. Dichloromethane (1ml) was then added, and the tubes vortexed vigorously. Followingcentrifugation to separate then two phases, the upper (aqueous) layerwas discarded. An aliquote (500 μl) of the lower, organic phase was thentransferred to a fresh tube containing 3 ml of 30 mM sodium boratebuffer (pH 9.0). Tubes were then vortexed and centrifuged once more. Themetabolite in the upper (aqueous) phase was then quantifiedfluorometrically, using excitation and emission wavelengths of 358 and458 nm, respectively, by reference to authentic standard (umbelliferone,Sigma).

Purification of Recombinant P450 and N-terminal Sequence Analysis

Expression from pCW/ompA-CYP3A4(His)₆ and pCW/ompA(+2)-CYP3A4(His)₆ wascarried out as previously described. Following lysozyme treatment of thecells and centrifugation, the spheroplasts were resuspended in bindingbuffer (20 mM potassium phosphate, pH 7.4, containing 500 mM potassiumchloride and 20% glycerol (v/v)) and stored at −70° C. Spheroplastsderiving from 125 ml culture were typically resuspended in 9 ml buffer.For P450 purification, spheroplasts were thawed on ice and sonicated inthe presence of the protease inhibitors aprotinin (1 μg/ml), leupeptin(1 μg/ml) and PMSF (1 mM), using a MSE SoniPrep 150 sonicator on 70%power. After centrifugation (1.2×10⁴ g, 12 min, 4° C.), the supernatantproteins were solubilised by stirring in the presence of Emulgen 911 (1mg/mg protein) at 4° C. for 60 min. The mixture was clarified bycentrifugation (10⁵ g, 60 min, 4° C. (and then loaded onto a Hi-Trapchelating column (Pharmacia) which had been charged with nickel ions andthen pre-equilibrated with binding buffer containing 0.10% Emulgen 911(w/v). The column was washed with a further ten column volumes ofbinding buffer (containing Emulgen 911), and then weakly-bindingproteins were removed with five column volumes of wash buffer (bindingbuffer containing 0.10% Emulgen 911 (w/v) and 75 mM imidazole). The redP450 band was eluted with elution buffer (binding buffer containing0.10% Emulgen 911 (w/v) and 1 M imidazole) —the imidazole concentrationwas kept high to elute the P450 into as small a volume as possible. Theprotein sample was dialysed overnight at 4° C. against several changesof anion-exchange buffer (20 mM Tris-Cl, pH 7.5, containing 20% glycerol(v/v), 0.2 mM dithiothreitol, 1 mM EDTA and 0.10% Emulgen 911 (w/v)),and then loaded onto a Hi-Trap Q column (Pharmacia). The flow-throughfraction, containing the P450, was then loaded onto an Econo-Pac® HTPcartridge (Bio-Rad) equilibrated with 10 mM sodium phosphate, pH 7.4,containing 20% glycerol (v/v), 1 mM EDTA, 1 mM DTT and 0.05% sodiumchelate (w/v). The concentration of sodium phosphate was increasedduring column washing to 25 mM, and then to 100 mM, with the P450eluting at 400 mM phosphate. The purity of the P450 preparation at eachstage of the procedure was assessed by SDS-polyacrylamide gelelectrophoresis, on 9% acrylamide (w/v) gels, straining with CoomassieBrilliant Blue R-250. The N-terminal sequencing, purified proteins werethen transferred onto Pro-blot polyvinylidene fluoride membrane (AppliedBiosystems) and stained. N-terminal amino acid sequence analysis wascarried out in the Department of Biochemistry, University of Dundee,using a Model 476A instrument (Applied Biosystems), with four to sixcycles of Edman degradation.

Results

Co-expression of OmpA-P450s with Reductase

As reported previously, the P450 expressed from pCW/ompA-CYP2D6 coupleswell with co-expressed reductase, catalysing typical CYP2D6-dependentactivities (see Example 2). Turnover measured for ompA-CYP2D6 were, ingeneral, slightly higher than those measured from the correspondingpCW/17α-CYP2D6 construct (data not shown).

We have since co-expressed a number of other ompA-P450s with reductasein E. coli, including CYP3A4 and CYP2A6. In contrast to CYP2D6, thesetwo ompA-P450s do not appear to couple as efficiently with reductase asthe corresponding 17α-P450s, since enzyme activities toward probesubstrates are generally lower. For example, coumarin 7-hydroxylateactivities in both cells and membranes are more than an order ofmagnitude lower with ompA-CYP2A6 compared with 17α-CYP2A6 (Table B).Similarly, for CYP3A4, testosterone 6β-hydroxylase activity in membranefractions is reduced with the ompA- construct (Table C). It isinteresting to note, however, that this difference between the twoconstructs is absent with a different probe substrate, nifedipine, usused (Table C). This emphasises the need to use several markeractivities, wherever possible.

TABLE B Yields and activities of CYP2A6 co-expressed with reducatse intwo plasmid system *Coumarin 7-hydroxylate activity Shocked cellsMembranes Cellular P450 yield Membrane P450 content (nmol/min/nmol(nmol/min/nmol P450 construct (nmol/l culture) (nmol/mg protein) P450)P450) 17α-CYP2A6 147 ± 10 0.19 ± 0.06 0.75 ± 0.17 2.9 ± 0.5 ompA-CYP2A641 0.17 <0.02 0.11 ompA(+2)-CYP2A6 137 ± 60 0.25 ± 0.11 0.29 ± 0.07 1.0± 0.2 Where possible, values are expressed as mean ± SD, based on atleast three independent determinations. Cytochrome P450 was quantifiedby Fe²⁺-CO vs. Fe²⁺ difference spectroscopy, in 100 mM Tris-HCl, pH 7.4,containing 20% (v/v) glycerol, 10 mM CHAPS and 1 mM EDTA. *Probeactivity for CYP2A6, measured fluorometrically.

In order to try and find an explanation for the relative lack ofcoupling of the ompA-P450s with reductase, six histidine residues wereappended to the C-terminus of the P450 expressed from pCW/ompA-CYP3A4.This allowed facile purification of the recombinant protein by nickelchelate affinity chromatography (described in Materials and Methods).This revealed that the ompA-P450 was not undergoing the expectedprocessing by bacterial signal peptidase, in that the signal peptide wasbeing retained. This may reflect the general level of over-expression ofthe P450, since it is known that the availability of signal peptidasecan be limiting, especially for hybrid precursors with low processingefficiencies (van Dijl et al. (1991) Mol. Gen. Genet. 227, 40-48).

Another factor which strongly influences signal peptidase activity isthe structure around the site of signal cleavage (Duffaud and Inoyue(1988) J. Biol. Chem. 263, 10224-10228; Barkocy-Gallagher et al. (1994)J. Biol. Chem. 269, 13609-13613), including the first few amino-acidsafter the signal peptide (Barkocy-Gallagher) and Bassford (1992) J.Biol. Chem. 267, 1231-1238; Nilsson and von Heijne (1992) FEBS Lett.299, 243-246). Over-expression of proteins with non-cleavable signalpeptides can completely block the translocation apparatus of the cell,leading to accumulation of protein precursors (Barkocy-Gallagher andBassford (1992) J. Biol. Chem. 267, 1231-1238). For our constructs, thesequence immediately following the signal peptide is the CYP3A4N-terminus. The amino-acid at position +1 relative to the site ofcleavage will therefore be methionine, whereas alanine is stronglypreferred in this position in bacterial genes (von Heijne (1986) Nucl.Acids Res. 14, 4683-4690).

We can therefore envisage three possible strategies for improving theprobability of signal peptide removal. Firstly, the bacterial signalpeptidase could be over-expressed from a separate, compatible plasmid,several of which have been described (van Dijl et al. (1991) Mol. Gen.Genet. 227, 40-48; Dalbey and Wickner (1985) J. Biol. Chem. 260,15925-15931; March and Inouye (1985) J. Biol. Chem. 260, 7206-7213). Asan alternative to, or perhaps in conjunction with, this first approach,it may also be advisable to introduce a short “linker” sequence betweenthe signal peptide and the P450, although there may be disadvantageswith producing a P450 with a short N-terminal extension. Finally, wecould try expression in a different bacterial strain, since this hasalso been shown to affect the extent of removal of signal peptides fromhybrid fusion proteins (Monteilhet et al. (1993) Gene 125, 223-228).

In order to try and circumvent this problem of signal retention, wetherefore decided to modify the cleavage site by introducing the firsttwo amino acids of the mature OmpA protein (-Ala-Pro-) between the OmpAleader and the N-terminus of the P450. This resulted in the constructspCW/ompA(+2)+CYP2A6 and pCW/ompA(+2)-CYP3A4 (described above). Incontrast to the ompA-P450s, these ompA(+2)-P450s appeared to couple moreefficiently with co-expressed reductase, resulting in higher substrateturnovers (Tables B and C). The recombinant protein expressed frompCW/ompA(+2)-CYP3A4(His)₆ was subsequently purified and subjected toN-terminal sequence analysis. This revealed that the protein was nowbeing correctly processed by bacterial signal peptidase, leading to theaccumulation in bacterial membranes of native CYP3A4 containing Ala-Pro-at the N-terminus.

TABLE C Yields and activities of CYP3A4 co-expressed with reductase intwo plasmid systems *P450 content *Reductase Cellular P450 yield(nmol/mg activity †Testosterone †Nifedipine P450 construct (nmol/lculture) protein) §(U/mg protein) 6β-hydroxylase oxidase 17α-CYP3A4 308± 43 0.36 ± 0.08 498 ± 30 4.9 ± 0.3  9.1 ± 0.7 ompA-CYP3A4 374 ± 23 0.49± 0.06 313 ± 12 2.5 ± 0.8 8.8 ompA-CYP3A4 292 ± 54 0.27 ± 0.05  535 ±112 9.9 ± 0.2 11.1 ± 0.3 Where possible, values are expressed as mean ±SD, based on at least three independent experiments. Cytochrome P450 wasquantified by Fe²⁺-CO vs. Fe²⁺ difference spectroscopy, in 100 mMTris-HCl, pH 7.4, containing 20% (v/v) glycerol, 10 mM CHAPS and 1 mMEDTA. *Measured in bacterial membrane fractions. §one unit of reductaseactivity is defined as 1 nmol of cytochrome c reduced per minute.†Metabolism of two known CYP3A4 substrates was assessed in membranefractions in the presence of 30 mM MgCl₂. Activities are expressed asturnovers in nmol formed per minute per nmol CYP3A4.

Summary

The straight fusion of the ompA leader sequence to P450s does not alwaysresult in a P450 isoenzyme which couples with coexpressed reductase.Changing the amino acid residues at the potential cleavage site of theompA sequence fused to the p450 sequence results in coupling andefficient removal of the leader sequence.

6 1 22 PRT Escherichia coli 1 Met Lys Tyr Leu Leu Pro Thr Ala Ala AlaGly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala 20 2 21 PRTEscherichia coli 2 Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu AlaGly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala 20 3 10 DNA Escherichia coli 3aggaggtcat 10 4 96 DNA Artificial Sequence Recombinant cDNA 4 atgaaatacctgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60 atggccatggatatcggatc cgaattccgc aacatg 96 5 34 DNA Artificial Sequence RecombinantcDNA 5 tcgacagccc gcctaatgag cgggcttttt ttta 34 6 63 DNA Escherichiacoli 6 atgaaaaaga cagctatcgc gattgcagtg gcactggctg gtttcgctac cgtagcgcag60 gcc 63

What is claimed is:
 1. A bacterial cell containing a functionalcytochrome P450 monooxygenase system, said cell comprising a geneticconstruct or constructs capable of expressing a cytochrome P450 andseparately a cytochrome P450 reductase wherein each of the cytochromeP450 and the cytochrome P450 reductase have at their N-terminus abacterial signal peptide.
 2. A bacterial cell according to claim 1wherein said cell is a bacterial cell of the family Enterobacteriaceae.3. A bacterial cell according to claim 2 wherein the cell is anEscherichia coli cell or a Salmonella typhimurium cell.
 4. A bacterialcell according to claim 1 wherein said cytochrome P450 is selected fromCYP1, CYP2, CYP3, or CYP4 families.
 5. A bacterial cell according toclaim 1 wherein said cytochrome P450 is selected from CYP1A, CYP1B,CYP2A, CYP2B, CYP2C, CYP2D, CYP2E, CYP3A, CYP4A, CYP4B and subfamiliesthereof.
 6. A bacterial cell according to claim 5 wherein the cytochromeP450 is selected from CYP3A4, CYP2D6, CYP2C9, CYP2D9, CYP2A6 and CYP2E1.7. A bacterial cell according to claim 1 wherein the cytochrome P450reductase is human cytochrome P450 reductase.
 8. A bacterial cellaccording to claim 1, wherein the bacterial signal peptide is selectedfrom an ompA, pelB, malE or phoA signal peptide.
 9. A bacterial cellaccording to claim 1 wherein the bacterial signal peptide on thecytochrome P450 is the same as the bacterial signal peptide on thecytochrome P450 reductase.
 10. A bacterial cell according to claim 1further comprising a genetic construct capable of expressing apolypeptide cofactor which aids the correct folding of the cytochromeP450 or the cytochrome P450 reductase, wherein the polypeptide cofactoris a molecular chaperone.
 11. A bacterial cell according to claim 1further comprising a genetic construct capable of expressing apolypeptide cofactor which aids transfer of electrons between thecytochrome P450 and the cytochrome P450 reductase.
 12. A bacterial cellaccording to claim 11 wherein said cofactor which aids transfer ofelectrons between the cytochrome P450 and the cytochrome P450 reductaseis cytochrome b₅ or the flavin mononucleotide domain of a cytochromeP450 reductase.
 13. A bacterial cell according to claim 1 furthercomprising a genetic construct capable of expressing any one of anenzyme capable of metabolising the product of a reaction catalysed bythe cytochrome P450 monooxygenase system.
 14. A bacterial cell accordingto claim 13 wherein said enzyme is selected from a glutathioneS-transferase, an epoxide hydrolase and a UDP-glucuronosyl transferase.15. A bacterial cell according to claim 1 wherein the cytochrome P450and the cytochrome P450 reductase are encoded by different geneticconstructs.
 16. A bacterial cell according to claim 1 wherein thecytochrome P450 and the cytochrome P450 reductase are encoded by thesame genetic construct.
 17. A bacterial cell according to claim 1wherein said cell is permeable to a compound which is a substrate of thecytochrome P450 monooxygenase system.
 18. A composition comprising aplurality of bacterial cells according to claim 1 wherein each cellcontains at least one genetic construct which encodes a cytochrome P450and a cytochrome P450 reductase.
 19. A method for converting a substractfor cytochrome P450 into a product, the method comprising admixing saidsubstrate with a bacterial cell which contains a functional cytochromeP450 monooxygenase system which can convert said substrate, wherein thecell comprises a genetic construct or constructs capable of expressing acytochrome P450 and separately a cytochrome P450 reductase wherein eachof the cytochrome P450 and the cytochrome P450 reductase have at theirN-terminus a bacterial signal peptide.
 20. The method according to claim19 wherein the bacterial signal peptide is any one of an ompA, pelB,malE or phoA signal peptides.
 21. The method according to claim 19wherein the bacterial cell is a bacterial cell of the familyEnterobacteriaceae.
 22. The method according to claim 19 wherein saidcytochrome P450 is as defined in claim
 4. 23. The method according toclaim 19 wherein said cytochrome P450 is as defined in claim
 5. 24. Thebacterial cell of claim 19 wherein said cytochrome P450 is as defined inclaim 6.